Rosoideae-specific duplication and functional diversification of FT-like genes in Rosaceae

© The Author(s) 2022. Published by Oxford University Press on behalf of Nanjing Agricultural University. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Horticulture Research, 2022, 9: uhac059


Dear Editor,
Rosaceae plants provide some of the most important fruits and f lowers, like apple, peach, pear, strawberry, and rose. Understanding the molecular genetic mechanisms that underlie the regulation of f lowering time, i.e. the transition from vegetative to reproductive growth, is therefore essential for securing f lower and fruit productivity. At least five pathways that regulate f lowering in model plants have been well characterized. Although components in the f lowering pathways may differ among species, most endogenous and exogenous cues are integrated into several key and conserved hubs, including the f lorigen that is encoded by FLOWERING LOCUS T (FT). Expressed in vasculature cells and transported to shoot apical meristems, FT interacts with the bZIP transcription factor FD and a scaffold protein 14-3-3 to form a florigen protein complex that induces the expression of inflorescence and f loral meristem genes. Because of its pivotal roles in f lowering time control and other developmental processes, the regulation of FT expression occurs at the transcriptional, post-transcriptional, and translational levels [1]. Gene copy-number variation via random/tandem duplication or whole genome duplication (WGD) accompanied by functional diversification provides another regulatory layer for FT function, and duplication of FT-like genes correlates tightly with crop domestication in rice, maize, and soybean [2][3][4]. However, this has not been investigated systematically in Rosaceae.
Here, we report the duplication and expression diversification of FT in rose, an emerging woody model for f lowering regulation. We identified two copies of FT via BLAST in the genomes of R. wichuraiana 'Basye's Thornless' (BT; RwFT1, Rw3g00010; and RwFT2, Rw0G011750) [5] and Rosa chinensis 'Old Blush' (OB; RcFT1, RchiOBHm_Chr3g0447431; and RcFT2, RchiOBHm_ Chr4g0439111) [6] (Fig. 1). An inter-and intra-genome macro-synteny analysis with MCScanX in five Rosaceae plants with chromosome-level genome assemblies revealed that the loci harboring rose FT1 and FT2 shared high levels of collinearity between OB and Rubus, Fragaria, Prunus, and Malus (Fig. 1A). A further detailed gene collinearity analysis demonstrated that the rose FT1 locus was syntenic to the FT1 loci of the other four Rosaceae plants (Fig. 1B). Despite the fact that the four genes up-and downstream of FT2 were highly collinear in all five Rosaceae species, no orthologous FT2 was detected in Prunus and Malus (Fig. 1C). Rose FT2 was also not listed in the syntenic gene pairs generated via the WGD event in Rosaceae [5]. A subsequent BLAST scan with reduced criteria in the genomes of these two species detected no sign of FT2-like sequences, implying that FT2like genes may have arisen from random duplication in Rosoideae or may have been lost in the common ancestor that gave rise to the Maloideae and Prunoideae lineages.
Comparison of rose FT1 and FT2 protein sequences with Arabidopsis FT and TSF demonstrated that rose FT2 differed from FT1 at 29 positions, with FT2 being seven amino acids longer than FT1. The two amino acids that distinguish FT (Tyr85 and Gln140) from TFL1 were conserved in both FT1 and FT2 (Fig. 1D). Detailed protein structure analyses demonstrated that both FT proteins contained the highly conserved PEBP domain. A further phylogeny reconstruction with FTlike proteins from twelve Rosaceae and two outgroup species (Ziziphus jujuba, Rhamnaceae, and Coptis chinensis, Ranunculaceae) identified two major clades (Fig. 1E). Clade I contained FT1-like genes from all 12 Rosaceae species and jujube, whereas clade II contained FT2-like genes from only Rosoideae (Rosa, Fragaria, and Rubus). Consistent with the recent WGD in Maloideae, Malus, Eriobotrya, and Pyrus have two FT1-like genes ( Figure 1E) [5]. Because no recent WGD event is present in the Rosoideae, these data indicate that a Rosoideae-specific duplication of FT2-like genes may have occurred prior to the separation of all Rosoideae plants from their common ancestor but after the separation of Rosoideae from Rosaceae (Fig. 1F). Interestingly, these FT2s were grouped together with the known rose FT and strawberry FvFT2 and FvFT3, which act as f lowering promoters [4,7]. However, the roles of rose FT1-like genes have never been investigated.
Therefore, we next compared the expression of the two FTs using transcriptome data from BT leaves harvested in November (non-f lowering season) and March (f lowering season) [8]. No reads were identified for either gene in leaves harvested in non-f lowering season, whereas a significant difference in expression was observed in leaves harvested during the f lowering season: RwFT2 expression was four times higher than that of RwFT1 (Fig. 1G). With a RT-qPCR approach, we next compared their expression in five tissues of BT, for which the f lowering had started (Fig. 1H). RwFT1 expression was about six-fold higher than that of RwFT2 in shoot apical tissues. On the other hand, RwFT2 expression was significantly higher than RwFT1 expression in both open leaves and f lower buds prior to anthesis. Both genes were expressed at similar levels in young leaves and young stems about 1 cm below the shoot apical tissues. These findings indicate that the two FTs have diverged in their expression. Consistent with this finding, a detailed examination of the 10 kb upstream of the translation initiation site showed significant variation in the numbers of potential cis-elements related to hormones (ABA and GA), circadian rhythm, and cold responses (Fig. 1I). In line with its relatively high expression in shoot apical tissues, the promoter of RwFT1 contained fifteen and eight cis-elements related to ABA and GA responses, whereas the RwFT2 promoter featured only six and three, respectively. The RwFT1 promoter harbored two cis-elements related to circadian rhythm regulation, whereas RwFT2 featured none. Conversely, the RwFT2 promoter had more cis-motifs related to cold temperature (nine) than RwFT1 (five).
In summary, we identified a new and Rosoideaespecific FT paralog generated from random duplication. As in strawberry, FT1 had diverged in sequence and expression pattern from the well-known FT2-like in rose [7]. Given the essential roles played by hormones in bud dormancy and f lowering time regulation in woody perennials [9,10], it is likely that rose FT1 serves as an important hub, integrating signals from hormones and the circadian clock as well as branching. Rose FT2 may play more roles in the response to cold stimulus. It is also possible that FT2 may regulate f lower bud development or f lower anthesis, possibilities that clearly await further investigation with additional molecular and genetic approaches.