Apple breeding is constrained by a juvenile phase of 5-12 years during which seedlings cannot flower. Yamagishi, Kishigami and Yoshikawa (2014) showed that inoculating germinating apple cotyledons with an Apple latent spherical virus (ALSV) vector simultaneously expressing Arabidopsis FLOWERING LOCUS T (AtFT) and silencing apple TERMINAL FLOWER1 (MdTFL1-1) induced flowering in over 90% of seedlings within 1.5-3 months and shortened the seed-to-seed life cycle to under a year, with no vector transmission to progeny in most cases. Paradoxically, apple's own FT orthologues MdFT1 and MdFT2 delivered from the same viral vector fail to induce flowering, even though stable transgenic MdFT1 overexpression flowers apple early, and a homologous-FT failure recurs in grapevine. Since 2014, the technique has been extended to pear, grapevine, strawberry, gentian, lisianthus, and citrus (with a different viral vector), and virus elimination and non-transmission to progeny have been demonstrated. However, the mechanistic question - why heterologous florigen succeeds where endogenous florigen fails in the viral-vector context, and how the FT/TFL1 balance sustains flowering - remains unresolved, the induced phenotype is transient in part of the plants, and application to established apple cultivars, Japanese pear, and Prunus remains undemonstrated. The problem is open.

Background

Fruit trees such as apple (Malus x domestica) pass through a juvenile phase lasting 5-12 years from germination before they can flower, which makes apple cross-breeding and genetic analysis extremely slow. The timing of flowering is controlled in part by florigen, a small mobile protein encoded by FLOWERING LOCUS T (FT) that is produced in leaves and transported to the shoot apical meristem, where it triggers the switch from vegetative to floral development. TERMINAL FLOWER1 (TFL1), a close relative of FT within the phosphatidylethanolamine-binding protein family, acts antagonistically to FT by repressing floral identity in the meristem, so the balance between FT and TFL1 activity determines when and where flowers form. Apple latent spherical virus (ALSV) is a mostly symptomless plant RNA virus that was repurposed as a viral vector: it can carry foreign gene fragments, so infecting a plant with an engineered ALSV construct can either express a transgene (here the Arabidopsis FT gene, AtFT) or, through virus-induced gene silencing (VIGS, the plant's antiviral RNA-degradation defense, which also suppresses matching host transcripts), repress an endogenous gene (here apple TFL1, MdTFL1-1). Yamagishi et al. (2011) showed that ALSV-AtFT alone makes apple seedlings flower within about two months of germination; Yamagishi, Kishigami and Yoshikawa (2014) then showed that combining AtFT expression with MdTFL1-1 silencing raises the flowering rate above 90%, compresses seed-to-seed generation time to under a year, and - because ALSV is usually not transmitted through seed - yields progeny free of the introduced construct, making the approach a transgene-free breeding accelerator ('virus-induced flowering', VIF). Two observations make the underlying mechanism nontrivial. First, stable transgenic overexpression of apple's own FT orthologue MdFT1 does induce early flowering in apple and poplar, and both apple FT paralogues (MdFT1, MdFT2) were characterized as functional florigen candidates (Kotoda et al. 2010; Tränkner et al. 2010); yet delivering MdFT1 or MdFT2 through the same ALSV vector does not induce apple flowering, with or without MdTFL1-1 silencing. Second, the same homologous-FT failure recurs in grapevine, where ALSV-VvFT does not induce flowering while ALSV-AtFT does (Maeda et al. 2020). In addition, the induced early-flowering state is transient in a fraction of plants, which revert to vegetative growth, and the inoculation protocol works only on cotyledons of germinating seedlings, not on established cultivars or rootstocks, which limits the technique to breeding programs that start from seed.

Problem Statement

The problem, as left open by Yamagishi, Kishigami and Yoshikawa (2014), is to establish the mechanistic basis and the applicability boundaries of ALSV-mediated virus-induced flowering in apple and other long-juvenile fruit trees. Concretely: (1) Mechanism of the induction and of the florigen-source asymmetry. Explain at the molecular-genetic level why concurrent expression of heterologous AtFT and silencing of MdTFL1-1 induces flowering in over 90% of apple seedlings, whereas expression of apple's own FT orthologues MdFT1 and MdFT2 from the same viral vector - even combined with MdTFL1-1 silencing - fails to induce flowering, despite stable transgenic MdFT1 overexpression being sufficient for early flowering in apple; identify the additional genetic factors or interactions (proposed to be specific to apple or to the FT/TFL1 pathway in Rosaceae) that distinguish effective from ineffective florigen sources, and clarify how the balance between FT activity and TFL1 repression sustains floral differentiation of the shoot apical meristem. The parallel failure of homologous FT in grapevine (VvFT with ALSV) invites an answer covering whether a common mechanism is at work. (2) Stability of the induced phenotype. The early/perpetual-flowering state is transient rather than stably fixed: in a fraction of seedlings the AtFT insert is lost and plants revert to vegetative growth. Explain this instability and determine what would make the phase transition persist. (3) Applicability envelope. Determine whether virus-induced flowering - with ALSV or an equivalent viral vector - can be extended from cotyledons of germinating seedlings to established apple cultivars and rootstocks, and to further long-juvenile fruit species where it is untested or has so far failed (for example Japanese pear, recalcitrant to systemic ALSV-AtFT/MdTFL1 infection, and Prunus species such as plum), while retaining the demonstrated property that progeny are free of the viral vector and of any introduced genetic construct. These components share one overarching objective: a mechanistically understood, stable, and broadly applicable transgene-free rapid-generation technique for fruit-tree breeding.

References

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