Orthologous PEBP-genes in C. pallens
Seven distinct homologues of PEBP-like gene family members were identified from the draft transcriptome of C. pallens (SRA accession number: GHUI00000000) (Samarth et al., 2019). The GIHRV-domain and LREY/HLHWIV-domain, both characteristic features of PEBP-family proteins were found to be conserved in the homologous sequences fromC. pallens (Fig. 1a).
The general topology of the reconstituted PEBP-protein family tree was consistent with the previous findings of Karlgren et al. (2011) and Liu et al. (2016), indicating that the PEBP phylogenetic tree can be sub-divided into three major clades. These involve MOTHER OF FT and TFL1 (MFT)-like, FLOWERING LOCUS T (FT)-like, and TERMINAL FLOWER1 (TFL1)-like (Fig. 1b). The PEBP-protein family tree was consistent with the species tree. Separate clades for homologous PEBP sequences belonging to monocots and dicots can be seen in the phylogenetic tree. All the C. pallens sequences were found to clade with PEBP protein sequences from species belonging to the Poaceae family. Of the seven contigs identified, one belongs to the MFT cluster, one belongs to the TFL1 group and five belong to the FT group (Fig. 1b). All sequences grouped with functionally categorised members of their respective families with high bootstrap values.
In C. pallens, five of the seven contigs which clustered within the FT group showed the presence of Tyr 85 residue, a critical amino acid for flower promoting activity. These contigs were named CpFT1-CpFT5, respectively (Fig. 1a). The external loop of FT (128-145 amino acid residues), critical for its florigenic activity (Ahn et al., 2006), was conserved in all the C. pallens FT sequences. All the FTs from C. pallens also had conserved characteristic features of an FT protein including His87, Glu109, Arg139, and Gln140 near the ligand-binding pocket. Structural modelling results indicate that Tyr85, His87, and Glu109 can form hydrogen bonds and interact with the Arg139 residue (Fig. S1). However, the Gln140 residue usually found in FT-like sequences has changed to His in CpFT3 and CpFT5, and to Pro in CpFT4 (Fig. 1a). The presence of proline at position 140 in CpFT4 may disturb the ligand-binding wall and may render the protein ineffective.
One of the C. pallens PEBP-like sequences clustering with the TFL1 group has both critical amino acid indicators of a TFL1-like sequence, a His88 and Asp140 (Fig. 1a). Segment B of exon 4 was also seen to be highly variable in C. pallens . During the genetic complementation test in the ft-1 mutant, CpFT1, CpFT2, CpFT3, CpFT4, CpFT5 and CpTFL1 were all found to accelerate flowering. All the FT/TFL1 -like genes significantly shortened the number of days for the complemented ft-1 plants to flower under inductive conditions relative to the mutant (Fig. 1c). This suggests that, in contrast to AtTFL1, which is a known floral repressor (Ahn et al., 2006), the C. pallens TFL1 -like gene promotes the floral transition. Therefore, from here on CpTFL1 is referred as the ANTI-TFL1 (CpATFL1 ). It is important to point out that genetic complementation assays have been widely used by the researchers to test the functionality of genes isolated from highly diverged species (such as gymnosperms) into model plants including Arabidopsis (Karlgren et al., 2011).