Characterization of FLOWERING LOCUS C Homologs in Apple as a Model for Fruit Trees

Abstract

To elucidate the molecular mechanism of juvenility and annual flowering of fruit trees, FLOWERING LOCUS C (FLC), an integrator of flowering signals, was investigated in apple as a model. We performed sequence and expression analyses and transgenic experiments related to juvenility with annual flowering to characterize the apple FLC homologs MdFLC. The phylogenetic tree analysis, which included other MADS-box genes, showed that both MdFLC1 and MdFLC3 belong to the same FLC group. MdFLC1c from one of the MdFLC1 splice variants and MdFLC3 contain the four conserved motives of an MIKC-type MADS protein. The mRNA of variants MdFLC1a and MdFLC1b contain intron sequences, and their deduced amino acid sequences lack K- and C-domains. The expression levels of MdFLC1a, MdFLC1b, and MdFLC1c decreased during the flowering induction period in a seasonal expression pattern in the adult trees, whereas the expression level of MdFLC3 did not decrease during that period. This suggests that MdFLC1 is involved in flowering induction in the annual growth cycle of adult trees. In apple seedlings, because phase change can be observed in individuals, seedlings can be used for analysis of expression during phase transition. The expression levels of MdFLC1b, MdFLC1c, and MdFLC3 were high during the juvenile phase and low during the transitional and adult phases. Because the expression pattern of MdFLC3 suggests that it plays a specific role in juvenility, MdFLC3 was subjected to functional analysis by transformation of Arabidopsis. The results revealed the function of MdFLC3 as a floral repressor. In addition, MdFT had CArG box-like sequences, putative targets for the suppression of flowering by MdFLC binding, in the introns and promoter regions. These results indicate that apple homologs of FLC, which might play a role upstream of the flowering signals, could be involved in juvenility as well as in annual flowering. Apples with sufficient genome-related information are useful as a model for studying phenomena unique to woody plants such as juvenility and annual flowering.

Keywords: FLOWERING LOCUS C; Malus domestica; Rosaceae; flowering; juvenility.

Figure 1

Amino acid sequence alignment of MdFLC and AtFLC. The MADS-, K-, I-, and C-domains are underlined. Identical amino acids for five and less proteins are shown in black and gray boxes, respectively.

Figure 2

A phylogenetic tree based on the amino acid sequences of FLC, SVP, AP1, SEP, and SOC1 homologs from various species. The tree was constructed by the neighbor-joining method after sequence alignment using the ClustalW program. Branch numbers refer to percentage of replicates that support the branch using the bootstrap method (1000 replicates). The scale bar corresponds to 0.1 amino acid substitutions per residue. The accession numbers of the proteins added to construct the phylogenetic tree are as follows: PtFLC (EU497676), VvFLC (GU133630), PpFLC (KP164015), BrFLC1 (DQ866874), BrFLC2, (DQ866875), BrFLC3 (DQ866876), MdJOINTLESS (DQ402055), AtSVP (AF211171), PsSVP (AY830919), PaJOINTLESS (EU332978), VvAP1 (GU133634), MdAP1 (EU672877), AtAP1 (BT004113), BdAP1 (HQ588324), MdMADS1 (U78947), MdMADS3 (U78949), MdMADS4 (U78950), MdMADS7 (AJ000760), MdMADS8 (AJ001681), FvSOC1 (FJ531999), MdSOC1 (DQ846833), GhSOC1 (JF701982), NtSOC1 (JQ686938), AtSOC1 (AY093967).

Figure 3

Seasonal changes in the expression levels of MdFLC1a (a), MdFLC1b (b), MdFLC1c (c), and MdFLC3 (d) in the leaves of adult trees. Total RNA was prepared for the expression analysis of MdFLC1 and MdFLC3 on May 1, June 1, July 1, and August 1, 2016. Relative expression was determined using triplicate measurements taken from three independent biological replicates. The relative expression levels were normalized against MdACTIN with standard errors, and the maximum level of the transcripts was set at 1.0. The values with different letters for each gene significantly differed between days at p < 0.05, according to a Tukey test.

Figure 4

Changes in the expression levels of MdFLC1b (a), MdFLC1c (b), and MdFLC3 (c) during phase transition in apple seedlings. The expression level of MdFLC1a was undetectable. Total RNA was prepared from the juvenile phase (A), transition phase (B and C), and adult phase (D and E) in early July 2009 as described in [27]. Relative expression was determined in triplicate measurements taken from three independent biological replicates. The relative expression levels were normalized against MdACTIN with standard errors, and the maximum level of the transcripts was set at 1.0. The values with different letters for each gene significantly differed between positions at p < 0.05 according to a Tukey test.

Figure 5

The overexpression of MdFLC3 and phenotypic analysis in Arabidopsis. Flowering phenotypes 32 days after sowing (a), days to bolting from sowing (b), the number of rosette leaves at bolting time (c), and the expression levels of AtFT (d) in the MdFLC3 transgenic (FOX1, 2) and wild-type (WT) plants grown under a 16 h photoperiod. The values with ** significantly differed between FOX and WT plants at p < 0.01, according to the Dunnett test. The values in (b,c) indicate means and standard errors (n = 30 or 31). The relative expression of AtFT was determined from triplicate measurements of three independent biological replicates 15 days after sowing (d). The relative expression levels were normalized against AtACTIN with standard errors and the maximum level of the transcripts was set at 1.0.

Figure 6

Transcript structures of MdFLC1 splice variants, MdFLC1a, MdFLC1b, and MdFLC1c. E and I with numbers indicate exons and introns, respectively. Numbers under the bar correspond to their sizes, in base pairs.