CpATFL1 acts as a promoter of flowering in C. pallens
The TFL1 protein is generally a floral repressor in annual and perennial plants, acting antagonistically to FT (Liu et al., 2016).CpATFL1, clustering with the TFL1 clade in the PEBP -gene family tree, also had the key conserved amino acid signatures of the TFL1 protein including His88, Asp144, and a variable segment B of exon 4 (Fig. 1a). However, functional analysis involving genetic complementation of the ft-1mutant of A. thaliana (Ler) with CpATFL1 indicated thatCpATFL1 was acting as a floral promoter: the ft-1 mutants complemented with the CpATFL1 gene flowered significantly earlier than the wild type A. thaliana plants. This response prompted a computational biology investigation of CpATFL1 . Sequence comparison of CpATFL1 with close relatives of FT in other Poaceae members including ZCN7 and ZCN8 (Lazakis, Coneva, & Colasanti, 2011) suggested that there is a conserved residue of Gln150 between two different gene sequences. Ho & Weigel (2014) showed that position 150-152 is conserved in FT -like sequences and is crucial for floral promoting activities (Fig. 4a, b ), whereas these positions are much more variable in TFL1 -like sequences. Position 150 in TFL1 -like sequences is generally occupied by either Glu or Asp as observed in ZCN1 and OsRCN4 , homologues of TFL1 in maize and rice respectively (Fig. 4b) (Ho & Weigel, 2014).
The change in Glu 150 to Gln 150 in CpATFL1 is caused by a single base-pair change from GAA to CAA. Glu is a negatively charged amino acid while Gln is a positively charged amino acid (Fig. 4c). Ho & Weigel (2014) showed that altering the surface charge of FT andTFL1 -like sequences can alter their corresponding activities. When analysed for the surface charge potential compared to ZCN8 (an FT homologue in maize) and ZCN1 (a TFL1 homologue in maize), CpATFL1 showed a similar charge distribution to ZCN8. The surface surrounding position 150 was strongly positive in ZCN8 and CpATFL1, whereas the surface around that position in ZCN1 was found to be highly negative (Fig. 4d). This change in the surface charge could be the reason for the flower promoting activity of CpATFL1, in contrast to the usual TFL1 activity.
Our gene expression studies also support the hypothesis thatCpATFL1 is associated with the induction of flowering in C. pallens (Fig. 2). Elevated expression of CpATFL1 was observed during the inductive summer condition in the leaf samples from the plants that subsequently flowered in all the transplants and plants growing at the control site (1070 m) in natural conditions. It is interesting to point out that generally expression of TFL1 -like proteins has been restricted to apical meristems (Liu et al.,2016). However, recent studies have shown that homologousTFL1 -like proteins are expressed in the leaves of a number of perennial plants including Arabis alpina, Rosa chinensis, Fragaria vesca, Jatropha curcus, Chrysanthemum morifolium and Manihot esculenta (Adeyemo, Hyde, & Setter, 2019; Gao et al., 2019; Iwata et al., 2012; Li et al., 2017; Wang et al., 2011). The specifics of the regulation of TFL1 in perennial plant species is still relatively unknown, except for A. alpina .