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 sub-divides
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 is 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. pallenssequences were found to clade with PEBP protein sequences from species
belonging to the Poaceae. 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 . In genetic
complementation of 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 to as theANTI-TFL1 (CpATFL1 ). It is important to point out that
genetic complementation assays using Arabidopsis mutants have been
widely used by researchers to test the functionality of genes isolated
from highly divergent species including gymnosperms (Karlgren et al.,
2011).