P. patens ssp. patens (strain Gransden 2004) were grown axenically on solid BCD-medium supplemented with 5 mM ammonium tartrate, 1 mM CaCl2 and 0.8 % (w/v) plant agar. The moss were grown in standard conditions, that was 24 light hour cycle, 25 °C and continuous photosynthetic photon flux of 155 µmoles m-2 sec-1. Protonema from P. patens was generated by homogenization of gametophyte colonies in 100 ml liquid supplemented BCD-medium by an Ultra-Turrax® T18 basic.
 

From the National Center for Biotechnology Information (NCBI), the protein sequences of four A. thaliana SR-proteins (SR30; SR34; SR34a; SR34b) were used as queries for homologous proteins in P. patens using NCBI BLASTp. The proteins from NCBI were built on pre V.1.6 gene models, so subsequently the proteins were used as queries in a BLASTp in P. patens database at http://www.phytozome.net to obtain V.1.6 gene models.To find expressed sequence tags (EST’s) in P. patens transcriptome BLAST at http://www.cosmoss.org was done. Genomic DNA was used as query in a BLASTn search in the pp0409_fil database. Multiple sequence alignment of EST’s was done using MUSCLE at the European Bioinformatics Institute (EBI).

SR-protein sequences from the SR-subfamily were aligned with ClustalW2 at EBI; the alignment was saved in the PHYLogeny Inference Package ( PHYLIP) format. A neighbor-joining tree was created using PHYLIP v.3.6.9 programs in order: Seqboot, Protdist, Neighbor and Consense all with default settings and with the option multiple datasets 100 when possible. A maximum likelihood tree was created using PhyML 3.0 online with 100 bootstraps. All phylogenetic trees were viewed in FigTree v.1.3.1.

RNA was isolated from 3 weeks old protonema and gametophyte tissue. RT-PCR was perfomed on three novel proteins and an endogenous control (beta-tubulin 1). Gene expression data were analyzed using ImageJ and one-way ANOVA with Tukey and Fisher method post-hoc analysis were performed.

RNA was isolated from 3 weeks old gametophytes. cDNA was synthesized and PCR using PpSR40-specific primers was performed. The PpSR40 cDNA was cloned into the pCMAK1 plasmid. Polyethylene glycol mediated transformation was performed. Transformants were selected for using zeocin for 2 weeks on selection, 2 weeks of selection and 2 weeks on selection. Overexpression transformants were confirmed by isolating RNA from 3 weeks old protonema and gametophyte tissue. RT-PCR was perfomed on three novel proteins and an endogenous control (beta-tubulin 1). Gene expression data were analyzed using ImageJ.