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 theft-1 mutant of Arabidopsis (Ler ) with CpATFL1 ,
indicated that CpATFL1 acted as a floral promoter: theft-1 mutants complemented with the CpATFL1 gene flowered
significantly earlier than the wild-type Arabidopsis. This response
prompted a computational biology investigation of CpATFL1 .
Sequence comparison of CpATFL1 with close relatives of FTin other Poaceae, including ZCN7 and ZCN8 of maize
(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 inFT -like sequences and is crucial for floral promoting activities
(Fig. 4a, b), whereas these positions are much more variable inTFL1 -like sequences. Position 150 in TFL1 -like sequences
is generally occupied by either Glu or Asp as observed in ZCN1and 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 (both RT-qPCR and RNA-seq) also support the
hypothesis that CpATFL1 is associated with the induction of
flowering in C. pallens (Fig. 2). Elevated expression ofCpATFL1 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 should be noted that, while it has been accepted that
expression of TFL1 -like proteins is restricted to apical
meristems (Liu et al., 2016), recent studies have shown that
homologous TFL1 -like proteins are expressed in the leaves of
several perennial plants, including Arabis alpine (Wang et al.,
2011), Rosa chinensis, Fragaria vesca (Iwata et al., 2012),
Jatropha curcus (Li et al., 2017), Chrysanthemum morifolium (Gao
et al., 2019) and Manihot esculenta (Adeyemo, Hyde, & Setter,
2019). The specifics of the regulation of TFL1 in perennial plant
species is still relatively unknown, except for A. alpina (Wang
et al., 2011).