Evolution of VRN2/Ghd7-Like Genes in Vernalization-Mediated Repression of Grass Flowering

Abstract

Flowering of many plant species is coordinated with seasonal environmental cues such as temperature and photoperiod. Vernalization provides competence to flower after prolonged cold exposure, and a vernalization requirement prevents flowering from occurring prior to winter. In winter wheat (Triticum aestivum) and barley (Hordeum vulgare), three genes VRN1, VRN2, and FT form a regulatory loop that regulates the initiation of flowering. Prior to cold exposure, VRN2 represses FT. During cold, VRN1 expression increases, resulting in the repression of VRN2, which in turn allows activation of FT during long days to induce flowering. Here, we test whether the circuitry of this regulatory loop is conserved across Pooideae, consistent with their niche transition from the tropics to the temperate zone. Our phylogenetic analyses of VRN2-like genes reveal a duplication event occurred before the diversification of the grasses that gave rise to a CO9 and VRN2/Ghd7 clade and support orthology between wheat/barley VRN2 and rice (Oryza sativa) Ghd7. Our Brachypodium distachyon VRN1 and VRN2 knockdown and overexpression experiments demonstrate functional conservation of grass VRN1 and VRN2 in the promotion and repression of flowering, respectively. However, expression analyses in a range of pooids demonstrate that the cold repression of VRN2 is unique to core Pooideae such as wheat and barley. Furthermore, VRN1 knockdown in B. distachyon demonstrates that the VRN1-mediated suppression of VRN2 is not conserved. Thus, the VRN1-VRN2 feature of the regulatory loop appears to have evolved late in the diversification of temperate grasses.The initiation of flowering is a major developmental transition in the plant life cycle. When flowering initiates, shoot apical meristems shift from forming vegetative organs such as leaves to forming flowers. In many plant species, flowering occurs at a particular time of year in response to the sensing of seasonal cues such as changes in day length and temperature. In some plants adapted to temperate climates, exposure to the prolonged cold of winter (vernalization) results in the ability to flower in the next growing season (Chouard, 1960;Amasino, 2010). Although vernalization ultimately enables flowering, vernalization responsiveness is typically an adaptation to ensure that flowering does not occur prematurely in the fall season. This has obvious adaptive value; for example, many vernalization-responsive plants become established in the fall season (during which flowering would not lead to successful reproduction) and then rapidly flower in the spring when conditions for reproduction and seed maturation are optimal.The grass family (Poaceae) originated approximately 70 million years ago as part of the tropical forest understory. However, grasses have since diversified across the globe occupying a variety of ecological niches (Kellogg, 2001). Exemplifying this, the ;3,800 species of grass subfamily Pooideae, including the economically important cereals wheat (Triticum aestivum, Triticeae), barley (...

Evidence for an Early Origin of Vernalization Responsiveness in Temperate Pooideae Grasses

“…Despite evidence for an early origin of vernalization responsiveness in Pooideae (this study), data suggest modifications to the underlying genetic pathway, at least at the base of the core Pooideae (Woods et al, 2016). In barley, VRN1 directly binds to the promoter of the flowering repressor VRN2, resulting in the downregulation of VRN2 during winter cold (Deng et al, 2015).…”

“…hypothesized to have played a key role in repeated plant diversification within the cold temperate zone (Ream et al, 2012;Preston and Sandve, 2013;Preston et al, 2016;Woods et al, 2016). One prediction of this hypothesis is the early origin of a vernalization-induced flowering gene network in predominantly temperate clades, facilitating their niche transition.…”

“…In barley, VRN1 directly binds to the promoter of the flowering repressor VRN2, resulting in the downregulation of VRN2 during winter cold (Deng et al, 2015). However, although it likely functions as a flowering repressor across grasses (Yan et al, 2004a;Weng et al, 2014;Nemoto et al, 2016;Woods et al, 2016), noncore Pooideae VRN2 transcripts are not downregulated in response to cold or VRN1 up-regulation (Woods et al, 2016). The co-option of VRN2 into the vernalization network might therefore have occurred through changes in the DNA-binding domain of VRN1 or through novel cis-regulatory changes in the VRN2 promoter.…”

“…The extent to which Pooideae species outside the core group plus Brachypodieae are responsive to vernalization is unclear (but see Woods et al, 2016). Indeed, although evidence suggests the late incorporation of VRN2 into the vernalization network at the base of core Pooideae (Woods et al, 2016), it remains to be determined whether other components of this gene network trace back earlier to the ancestor of Pooideae.…”

“…For example, disruption of the regulatory site in the first intron or CArG-like motif of the wheat and barley VRN1 promoter results in constitutive expression of VRN1, leading to early flowering in the absence of cold (Yan et al, 2003(Yan et al, , 2004bvon Zitzewitz et al, 2005;Cockram et al, 2007;Pidal et al, 2009). The extent to which Pooideae species outside the core group plus Brachypodieae are responsive to vernalization is unclear (but see Woods et al, 2016). Indeed, although evidence suggests the late incorporation of VRN2 into the vernalization network at the base of core Pooideae (Woods et al, 2016), it remains to be determined whether other components of this gene network trace back earlier to the ancestor of Pooideae.…”