Phylogenetic, structural and functional analysis of the
PEBP-sequences in C. pallens
There is no published genome for Chionochloa. All 23 New ZealandChionochloa species have the same chromosome number (n=7) and are
regarded as ancient hexaploids (2n = 6x = 42) which have undergone
diploidization (Linder & Barker, 2014; Murray, De Lange, & Ferguson,
2005). The reference ‘C. pallens’ transcriptome assembly
published in Samarth et al. (2019) was used to identify the potential
homologues of the PEBP protein family (Table 2). The identified
homologous PEBP sequences were further clustered into their respective
sub-families using a phylogenetic approach. The PEBP-phylogenetic tree
consisting of 49 protein sequences (Table S1) was reconstructed using
the maximum likelihood method with Jukes Cantor substitution matrix and
1000 bootstrap replication using the PhyML package. Homologous PEBP
protein sequences were also modelled using a homology modelling approach
at the SWISS-PROT server and visualised using Pymol (student version).
For functional analysis, the homologous PEBP-gene sequences
(CpFT1, CpFT2, CpFT3, CpFT4, CpFT5 and CpTFL1 ) were
transformed into Arabidopsis accession Landsberg erecta ft-1mutant plants. The cDNA sequences were cloned into the binary vector
(pB2GW7), downstream of the AtSUC2 promoter, and transformed intoAgrobacterium tumefaciens GV3101 by electroporation. Arabidopsisft-1 mutants were transformed using the method of
Martinez-Trujillo et al., (2004) as follows: A. tumefaciensharbouring the binary vector were streaked onto LB media plates with
selective antibiotics and incubated at 28 ºC for two days. The bacteria
were then scraped off the plate and resuspended in 10 mL of infiltration
media (0.5x MS (Duchefa), 0.05% Silwett L-77 (Lehle Seeds), 5%
sucrose) to an OD600 = 2 and drops of this solution were pipetted onto
unopened flower buds of Arabidopsis. This was repeated three times at
2–3-day intervals. The resulting seed was sown on seed-raising mix and
transformants selected by spraying the young seedlings with Basta
herbicide (glufosinate ammonium; Bayer) at 120 mg/ L. The number of
rosette leaves was counted at the time of flowering. Wild-type
Arabidopsis Landsberg erecta plants flower after producing
~ 8-10 leaves whereas ft-1 is a late-flowering
mutant, flowering after ~20 leaves (Martinezzapater &
Somerville, 1990).