Apple (Malus × domestica) homologs of the Arabidopsis floral repressor FLOWERING LOCUS C (FLC) — MdFLC1, with three splice variants, and MdFLC3 — were shown by Kagaya et al. (2020) to be expressed during flowering induction and the juvenile-to-adult phase transition, with MdFLC3 acting as a floral repressor in Arabidopsis; however, every functional claim still rests on expression correlations plus a single heterologous overexpression. The open problem is to determine, by direct experiment, whether MdFLC1 and MdFLC3 actually regulate juvenility and annual flowering (the recurring flowering cycle of adult apple trees) in apple: whether MdFLC1 has a direct physiological role, whether MdFLC3's juvenility-associated expression is independent of seedling genotype, whether MdFLC proteins bind the CArG box-like elements of the apple FT homologs MdFT1 and MdFT2 to regulate them, and whether MdFLC characteristics correlate quantitatively with the length of the juvenile period, and hence whether they could serve as breeding markers for short-juvenile-phase cultivars. No resolution of these four linked questions is known in the literature to date.

Background

Apple, like most fruit trees, passes through a long juvenile phase — typically several years of vegetative growth — before it can flower and set fruit. This juvenile period is the main bottleneck in apple breeding, because every cross requires the progeny to reach reproductive maturity before it can be evaluated and crossed again. Understanding which genes control the length of the juvenile phase is therefore both a fundamental question in the developmental biology of woody perennials and a practical target for marker-assisted selection, in which breeders use DNA- or expression-based markers to screen seedlings for a trait years before it appears.

Much of the conceptual framework comes from Arabidopsis thaliana, an annual plant in which flowering time is controlled by a well-mapped gene network. A central player is FLOWERING LOCUS C (FLC), a MADS-box transcription factor (transcription regulators carrying a conserved DNA-binding MADS domain) that represses flowering; it is epigenetically silenced by prolonged winter cold (vernalization). FLC represses the floral integrators FT (FLOWERING LOCUS T, encoding the mobile flowering signal "florigen") and SOC1 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1, another floral-integrator gene) by binding, as part of a protein complex, to CArG boxes — consensus CC(A/T)₆GG-type MADS-binding motifs — in their chromatin (Helliwell et al. 2006).

Apple contains FLC-like MADS-box genes, identified genome-wide by Kumar et al. (2016). Kagaya et al. (2020) characterized two of them as the focus of apple FLC research. MdFLC1 (gene model MD05G1037100) produces three splice variants — MdFLC1a and MdFLC1b, whose retained introns interrupt the reading frame so the predicted proteins lack the K- and C-domains required for interactions of MIKC-type MADS proteins (MIKC denotes the conserved MADS-, I-, K-, and C-domain architecture of plant MADS transcription factors), and the full-length MdFLC1c; MdFLC3 (MD10G1041100) encodes a complete MIKC-type MADS protein. In adult trees, MdFLC1 transcript levels fall during the period of flowering induction, resembling the seasonal behavior of the known apple floral repressor MdTFL1 (the apple homolog of TERMINAL FLOWER 1, which maintains vegetative identity in shoot tips), while MdFLC3 does not. In seedlings — where the juvenile, transitional, and adult phases can be tracked within single individuals — MdFLC1b, MdFLC1c, and MdFLC3 are all highly expressed in the juvenile phase and low in the transitional and adult phases. Overexpression of MdFLC3 in Arabidopsis delayed flowering, identifying it as a floral repressor in that heterologous system; MdFLC1 was never functionally tested. Sequence inspection of the two apple FT homologs, MdFT1 and MdFT2, revealed CArG box-like elements in their promoter and intron regions, suggesting — but not demonstrating — direct regulation of MdFT by MdFLC proteins, as in the Arabidopsis FLC–FT module.

Two adjacent results frame the question. First, Zong et al. (2019) reported that annotated apple FLC-like proteins group phylogenetically closer to Arabidopsis AP1/CAL-type MADS factors than to FLC, and that constitutive expression of an apple FLC3-like gene in blueberry promoted (rather than repressed) flowering; the FLC-clade identity and functional direction of apple FLC-like genes is therefore not fully settled, making direct native-species evidence especially valuable. Second, genome-wide association studies of flowering date in apple (Urrestarazu et al. 2017) have mapped loci for flowering and ripening period, but no genetic study has connected MdFLC genes to the length of the juvenile phase. What is missing is direct experimental evidence of what the MdFLC genes do in apple itself, and whether they can be used as quantitative breeding markers for juvenility.

Problem Statement

Establish, by direct experimental evidence in apple, whether the FLOWERING LOCUS C homologs MdFLC1 and MdFLC3 regulate juvenility and annual flowering, and whether they can be used as breeding markers for the length of the juvenile phase. Concretely: (1) determine the physiological role of MdFLC1 during apple development directly, beyond its expression pattern (its splice variants were characterized only at the sequence and expression level, and no functional test of MdFLC1 exists); (2) confirm that the juvenility-associated expression of MdFLC3 is not an artifact of the specific crossed seedlings used, by analyzing additional genotype combinations and/or apomictic (genotype-fixed) seedlings; (3) test whether MdFLC1 and MdFLC3 proteins bind the CArG box-like sequences in the promoter and intron regions of MdFT1 and MdFT2 and thereby regulate MdFT, as the Arabidopsis FLC-FT module would predict, rather than these remaining merely candidate binding sites; and (4) determine quantitatively whether MdFLC characteristics (expression levels, splice-variant composition, or allelic variation) correlate with the length of the juvenile period across genotypes, and hence whether MdFLC genes could serve as markers for marker-assisted selection of short-juvenile-phase apple cultivars, a question to be settled rather than a presupposed outcome. These four questions serve one objective — establishing the function of MdFLC genes in apple juvenility and their breeding relevance — and each is posed interrogatively: rigorous experimental evidence that MdFLC1 or MdFLC3 does not regulate juvenility or annual flowering in apple, or that MdFLC characteristics are uncorrelated with juvenile-period length, resolves the corresponding question as definitively as positive evidence. The four questions all bear on this one objective, and in question (4) the quantitative correlation is the determination, with marker utility its consequent.

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