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Pre-mRNA alternative splicing is a conserved mechanism for eukaryotic cells to leverage existing genetic resources to create a diverse pool of protein products. It is regulated in coordination with other events in RNA metabolism such as transcription, polyadenylation, RNA transport, and nonsense-mediated decay  via  protein networks. SERINE/ARGININE-RICH 45 (SR45) is thought to be a neutral splicing regulator. It is orthologous to a component of the apoptosis and splicing-associated protein (ASAP) complex functioning to regulate RNA metabolism at multiple levels. Within this context, we try to understand why the  sr45-1  mutant Arabidopsis has malformed flowers, delayed flowering time, and increased disease resistance. Prior studies revealed increased expression for some disease resistance genes and the flowering suppressor  Flowering Locus C  ( FLC ) in  sr45-1  mutants and a physical association between SR45 and reproductive process-related RNAs. Here, we used Tandem Mass Tag-based quantitative mass spectrometry to compare the protein abundance from inflorescence between Arabidopsis wild-type (Col-0) and  sr45-1  mutant plants. A total of 7,206 proteins were quantified, of which 227 proteins exhibited significantly different accumulation. Only a small percentage of these proteins overlapped with the dataset of RNAs with altered expression. The proteomics results revealed that the  sr45-1  mutant had increased amounts of enzymes for glucosinolate biosynthesis which are important for disease resistance. Furthermore, the mutant inflorescence had a drastically reduced amount of the Sin3-associated protein 18 (SAP18), a second ASAP complex component, despite no significant reduction in  SAP18  RNA. The third ASAP component protein, ACINUS, also had lower abundance without significant RNA changes in the  sr45-1  mutant. To test the effect of SR45 on SAP18, a SAP18-GFP fusion protein was overproduced in transgenic Arabidopsis Col-0 and  sr45-1  plants. SAP18-GFP has less accumulation in the nucleus, the site of activity for the ASAP complex, without SR45. Furthermore, transgenic  sr45-1  mutants overproducing SAP18-GFP expressed even more  FLC  and had a more severe flowering delay than non-transgenic  sr45-1  mutants. These results suggest that SR45 is required to maintain the wild-type level of SAP18 protein accumulation in the nucleus and that  FLC -regulated flowering time is regulated by the correct expression and localization of the ASAP complex.

frontiers in plant science

Quantitative Proteomics Reveals a Role for SERINE/ARGININE-Rich 45 in Regulating RNA Metabolism and Modulating Transcriptional Suppression via the ASAP Complex in Arabidopsis thaliana