Evolution of VRN2/Ghd7-Like Genes in Vernalization-Mediated Repression of Grass Flowering1[OPEN] Daniel P. Woods2,MeghanA.McKeown2, Yinxin Dong, Jill C. Preston, and Richard M. Amasino* Laboratory of Genetics, U.S. Department of Energy Great Lakes Bioenergy Research Center (D.P.W., R.M.A.), and Department of Biochemistry (D.P.W., Y.D., R.M.A.), University of Wisconsin, Madison, Wisconsin 53706; Department of Plant Biology, University of Vermont, Burlington, Vermont 05405 (M.A.M., J.C.P.); and College of Horticulture, Northwest A&F University, Yangling, Shaanxi 712100, People’s Republic of China (Y.D.) ORCID IDs: 0000-0002-1498-5707 (D.P.W.); 0000-0002-0187-4135 (Y.D.); 0000-0002-9211-5061 (J.C.P.); 0000-0003-3068-5402 (R.M.A.). Flowering of many plant species is coordinated with seasonal environmentalcuessuchastemperatureand 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)andbarley(Hordeum vulgare), three genes VRN1, VRN2,andFT 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.OurBrachypodium 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 response to the sensing of seasonal cues such as changes transition in the plant life cycle. When flowering initiates, in day length and temperature. In some plants adapted to shoot apical meristems shift from forming vegetative or- temperate climates, exposure to the prolonged cold of gans such as leaves to forming flowers. In many plant winter (vernalization) results in the ability to flower in species, flowering occurs at a particular time of year in the next growing season (Chouard, 1960; Amasino, 2010). Although vernalization ultimately enables flowering, vernalization responsiveness is typically an adaptation 1 J.C.P. was supported by USDA-HATCH and by the National Sci- to ensure that flowering does not occur prematurely in the ence Foundation (IOS-1353056). R.M.A. was supported by the National fall season. This has obvious adaptive value; for example, Science Foundation (Grant IOS-1258126) and by the Great Lakes Bio- many vernalization-responsive plants become established energy Research Center (Department of Energy Biological and Environ- in the fall season (during which flowering would not lead fi mental Research Of ce of Science Grant DE-FCO2-07ER64494). D.P.W. to successful reproduction) and then rapidly flower in the was supported in part by a National Institutes of Health-sponsored spring when conditions for reproduction and seed mat- predoctoral training fellowship to the University of Wisconsin Genetics Training Program. Y.D. was funded by the China Scholarship Council. uration are optimal. 2 These authors contributed equally to the article. The grass family (Poaceae) originated approximately * Address correspondence to [email protected]. 70 million years ago as part of the tropical forest under- The author responsible for distribution of materials integral to the story. However, grasses have since diversified across the findings presented in this article in accordance with the policy de- globe occupying a variety of ecological niches (Kellogg, scribed in the Instructions for Authors (www.plantphysiol.org) is: 2001). Exemplifying this, the ;3,800 species of grass Richard M. Amasino ([email protected]). subfamily Pooideae, including the economically impor- D.P.W., M.A.M., J.C.P., and R.M.A. conceived and designed re- tant cereals wheat (Triticum aestivum,Triticeae),barley search plans; M.A.M. performed experiments pertaining to Figures (Hordeum vulgare, Triticeae), oat (Avena sativa, Poeae), and 1 to 3 and associated supplemental alignment advised by J.C.P.; rye (Lolium perenne, Poeae), have adapted to cool climates D.P.W. and Y.D. performed experiments pertaining to Figures 3 to 6 and associated supplemental figures advised by R.M.A.; D.P.W. of both northern and southern hemispheres (Hartley, and M.A.M. wrote the article with contributions of all authors; J.C.P. 1973; Grass Phylogeny Working Group, 2001; Edwards and R.M.A. supervised and complemented the writing. and Smith, 2010). Phylogenetic analyses indicate the [OPEN] Articles can be viewed without a subscription. above-mentioned species within Triticeae and Poeae are www.plantphysiol.org/cgi/doi/10.1104/pp.15.01279 a closely related group often referred to as crown or core Ò 2124 Plant Physiology , April 2016, Vol. 170, pp. 2124–2135, www.plantphysiol.org Ó 2016 American Society of Plant Biologists. All Rights Reserved. Downloaded from www.plantphysiol.org on August 17, 2016 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Evolution of VRN2/Ghd7 in Grasses pooid grasses (Schneider et al., 2009; Grass Phylogeny FT also requires long days to become activated; thus, Working Group II, 2012). The remaining noncore pooids flowering only occurs during the lengthening days of are in tribes consecutively sister to the core pooids, in- spring and summer (Yan et al., 2006; Hemming et al., cluding Brachypodieae that contains the emerging plant 2008; Sasani et al., 2009). In wheat and barley, VRN2 is model Brachypodium distachyon (Meliceae and Stipeae; necessary for the vernalization requirement because Brkljacic et al., 2011; Grass Phylogeny Working Group II, deletions of the entire locus or point mutations in the 2012; Fig. 1). CCT domain result in spring varieties, which do not It is hypothesized that vernalization responsiveness require vernalization (Yan et al., 2004; Dubcovsky et al., evolved early during the diversification of Pooideae, as 2005; Karsai et al., 2005; von Zitzewitz et al., 2005; a key adaptation allowing for their transition into the Distelfeld et al., 2009b). temperate zone (Preston and Sandve, 2013; Fjellheim In wheat, there is a negative correlation between et al., 2014). Within core Pooideae, many species have VRN1 and VRN2 expression in leaves. VRN1 levels been characterized as vernalization responsive (Heide, increase during cold and remain elevated following 1994; Grass Phylogeny Working Group, 2001; Grass cold (Trevaskis et al., 2003; Yan et al., 2003; Sasani et al., Phylogeny Working Group II, 2012). However, it is 2009), and this correlates with the stable reduction of unclear how widespread vernalization responsiveness VRN2 during and after cold exposure (Yan et al., 2004). is outside core pooids and whether pooids with this Recently, it was shown that VRN1 binds to the VRN2 trait share a conserved ancestral vernalization path- promoter and thus directly regulates VRN2 expression way. To explore the extent to which the vernalization (Deng et al., 2015). Furthermore, mutations in the wheat pathway is conserved in Pooideae, we characterized the VRN1 locus result in elevated VRN2 expression and expression and function of vernalization pathway ho- delayed flowering (Chen and Dubcovsky, 2012). The mologs in B. distachyon and other pooids. delayed flowering phenotype in the vrn1 mutants is The current molecular model of vernalization re- largely due to the presence of VRN2 because wheat vrn1 sponsiveness in wheat and barley involves a leaf-specific vrn2 double mutants flower significantly earlier than regulatory loop among VERNALIZATION1 (VRN1), vrn1 single mutants (Chen and Dubcovsky, 2012). VRN2,andVRN3 (Dennis and Peacock, 2009; Distelfeld Expression of VRN1 in the noncore pooid B. distachyon et al., 2009a; Greenup et al., 2009; Sasani et al., 2009), is consistent with it being conserved as floral promoter the latter of which is homologous to Arabidopsis involved in vernalization (Ream et al., 2014; for review (Arabidopsis thaliana) FT, which encodes a small protein on flowering in B. distachyon, see Woods and Amasino that moves from leaves to the shoot apical meristem to 2015). As in wheat and barley, B. distachyon VRN1 promote flowering (Yan et al., 2006; Turck et al., 2008). (BdVRN1) mRNA levels increase quantitatively during During growth of vernalization-requiring cereals in the increasing durations of cold exposure and remain ele- fall season, the CONSTANS-like gene VRN2 represses vated post cold (Ream et al., 2014; Woods et al., 2014). FT to prevent flowering, and the FRUITFULL-like gene Furthermore, overexpression of BdVRN1 results in rapid VRN1 is transcribed at very low levels (Yan et al., 2004a, flowering and is correlated with elevated BdFT and re- 2006; Hemming et al., 2008; Sasani et al., 2009). During duced BdVRN2 expression (Ream et al., 2014). However, winter, VRN1 transcript levels increase, causing the contrary to VRN2 behavior in core pooids, BdVRN2 repression of VRN2 and the derepression of VRN3/FT mRNA levels increase rather than decrease during cold, (Yan et al., 2004b; Trevaskis et al., 2006; Sasani et al., despite a simultaneous increase in BdVRN1 expression 2009). Although vernalization alleviates FT repression, (Ream et al., 2014). Moreover, after cold exposure, BdVRN2 expression levels return to prevernalization expression levels.