Winter wheat must experience prolonged cold (vernalization) before it can flower; each new generation must re-experience winter because the parental 'memory of winter cold' is erased during seed development. Niu et al. (Nature Plants, 2024) established a two-step reset mechanism: cold-induced activation of the floral promoter gene TaVRN1, inherited by early embryos, is switched off during embryogenesis (involving restoration of the repressive histone mark H3K27me3 and the BAH-domain reader protein TaRVR1), while the floral repressor gene TaVRN2, silenced during seed development, is rapidly reactivated by light in the germinating embryo. The molecular machinery executing each step, however, remains unidentified: the histone methyltransferases that causally deposit the active marks H3K4me3 and H3K36me3 at TaVRN1, the embryo-specific transcription factors that cooperate with TaRVR1 to silence TaVRN1, and the photoreceptors and signaling components that mediate light-induced TaVRN2 reactivation. This problem asks for the identification and functional validation of these components, completing the causal mechanism of how each wheat generation's requirement for winter cold is restored.

Background

Vernalization is the acquisition, after prolonged exposure to winter cold, of the competence to flower in spring; it prevents temperate plants from flowering before winter has passed. In wheat (Triticum aestivum), flowering is governed by three central genes: VERNALIZATION 1 (TaVRN1, an APETALA1-like MADS-box transcription factor and potent floral promoter), VERNALIZATION 2 (TaVRN2, a zinc-finger CCT-domain floral repressor), and VERNALIZATION 3 (TaVRN3, the wheat ortholog of FLOWERING LOCUS T encoding the mobile flowering hormone florigen). Before cold, TaVRN2 represses TaVRN3; prolonged cold represses TaVRN2 and activates TaVRN1, which then maintains TaVRN2 repression in a stabilizing feedback loop, and this active TaVRN1 state persists through post-cold vegetative growth as an epigenetic 'memory of winter cold'.

Gene activity in this process is tightly coupled to histone modifications — methyl groups attached to specific lysines of histone H3 that mark chromatin as active or repressive. Trimethylation of H3 lysine 4 (H3K4me3, enriched at promoters of active genes) and H3 lysine 36 (H3K36me3, enriched in bodies of active genes) are associated with active transcription, whereas trimethylation of H3 lysine 27 (H3K27me3), deposited by Polycomb repressive complex 2 (PRC2, a conserved histone methyltransferase complex), is associated with stable gene silencing. H3K4me3 is deposited by COMPASS-like methyltransferase complexes (named after the yeast/bacterial Set1/COMPASS family), and H3K36me3 by methyltransferases of the SDG8/EFS (EARLY FLOWERING IN SHORT DAYS / SET DOMAIN GROUP 8) type. Proteins that bind specific histone marks and translate them into regulatory outcomes are called chromatin readers; one class of H3K27me3 readers contains a BAH (bromo-adjacent homology) domain.

Niu et al. (Nature Plants 10:37–52, 2024) showed how the cold memory is reset across generations in the winter wheat cultivar KN9204. Cold-induced TaVRN1 activation is transmitted through gametes to the zygote and early embryos, but TaVRN1 is switched off during subsequent embryogenesis. At the chromatin level, non-vernalized seedlings carry H3K27me3 at TaVRN1; after six weeks of cold this repressive mark is largely lost, H3K4me3 accumulates at the promoter and 5' region and H3K36me3 in the gene body, and this configuration is maintained after cold; during embryo development the active marks are removed and H3K27me3 is restored. A wheat BAH-domain H3K27me3 reader, named TaRVR1 (REPRESSOR OF VERNALIZATION 1, homologous to the Arabidopsis H3K27me3 reader AIPP3 and to Brachypodium BdRVR1), is required for this resetting: CRISPR/Cas9 mutants disrupting all three TaRVR1 homeoalleles (one per each of the A, B and D subgenomes of hexaploid wheat) derepress TaVRN1, flower rapidly without vernalization, and fail to silence TaVRN1 during embryo development. In parallel, TaVRN2 stays silent throughout grain and embryo development but is reactivated by light within hours of germination — in dark-grown seedlings, TaVRN2 mRNA is detectable within 30 minutes of illumination, and reactivation is triggered by blue, red and far-red light across a wide intensity range — restoring repression of TaVRN3 so that the filial plant again requires winter cold. Analogous dynamics occur in the diploid progenitor Triticum urartu.

The framework is therefore established, but its executors are not. TaRVR1's H3K27me3 binding is inferred from homology and structural modeling, without direct in vitro binding assays, and no wheat COMPASS-like H3K4 methyltransferase has been functionally implicated at TaVRN1. Known cold-responsive regulators of TaVRN1 — the transcription factor TaRF2b acting with the vernalization-induced long noncoding RNA VAS (Xu et al. 2021), and the SHORT VEGETATIVE PHASE (SVP)-like MADS-box factor TaVRT2 (Xie et al. 2021) — have not been connected to TaRVR1 or to the chromatin modifiers. The embryo- or seed-specific transcription factors that cooperate with TaRVR1 to switch TaVRN1 off are unknown, and the photoreceptors and signaling pathway mediating light-triggered TaVRN2 reactivation are unidentified.

Problem Statement

Identify, and causally validate, the molecular machinery that executes the vernalization-memory reset cycle in winter wheat, left open by the two-step reset mechanism established by Niu et al. (Nature Plants, 2024): (1) determine whether and how wheat COMPASS-like H3K4 methyltransferases and the EFS-like H3K36 methyltransferase deposit H3K4me3 and H3K36me3 at TaVRN1, and how this deposition drives transcriptional initiation, elongation or splicing during cold induction and post-cold maintenance of TaVRN1 (the post-cold maintenance role of H3K36me3 via TaEFS/TaSDG8 has been established by Liu et al. 2024; the cold-induction phase and the H3K4me3/COMPASS arm remain open); (2) determine whether and how cold-responsive transcriptional regulators — including but not limited to TaRF2b, TaVRT2 and vernalization-induced noncoding RNAs such as VAS — act with TaRVR1 and chromatin modifiers during cold induction and maintenance of TaVRN1 expression; (3) identify the embryo- or seed-specific transcription factor(s) and their mechanism of cooperation with TaRVR1 and repressive chromatin machinery to switch off TaVRN1 during embryogenesis; and (4) elucidate the molecular pathway by which light rapidly reactivates TaVRN2 in the sprouting embryo — the photoreceptors and signaling components involved. The four parts are jointly required and together constitute the objective: each concerns a distinct step of the reset cycle, and each step's mechanism requires a causal role for its components, not merely a correlation between chromatin state or expression pattern and the reset cycle.

References

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