ODDSOC2 is a MADS box floral repressor that is down-regulated by vernalization in temperate cereals

Abstract

In temperate cereals, such as wheat (Triticum aestivum) and barley (Hordeum vulgare), the transition to reproductive development can be accelerated by prolonged exposure to cold (vernalization). We examined the role of the grass-specific MADS box gene ODDSOC2 (OS2) in the vernalization response in cereals. The barley OS2 gene (HvOS2) is expressed in leaves and shoot apices but is repressed by vernalization. Vernalization represses OS2 independently of VERNALIZATION1 (VRN1) in a VRN1 deletion mutant of einkorn wheat (Triticum monococcum), but VRN1 is required to maintain down-regulation of OS2 in vernalized plants. Furthermore, barleys that carry active alleles of the VRN1 gene (HvVRN1) have reduced expression of HvOS2, suggesting that HvVRN1 down-regulates HvOS2 during development. Overexpression of HvOS2 delayed flowering and reduced spike, stem, and leaf length in transgenic barley plants. Plants overexpressing HvOS2 showed reduced expression of barley homologs of the Arabidopsis (Arabidopsis thaliana) gene FLOWERING PROMOTING FACTOR1 (FPF1) and increased expression of RNase-S-like genes. FPF1 promotes floral development and enhances cell elongation, so down-regulation of FPF1-like genes might explain the phenotypes of HvOS2 overexpression lines. We present an extended model of the genetic pathways controlling vernalization-induced flowering in cereals, which describes the regulatory relationships between VRN1, OS2, and FPF1-like genes. Overall, these findings highlight differences and similarities between the vernalization responses of temperate cereals and the model plant Arabidopsis.

Figure 1.

HvOS1 and HvOS2 are members of a grass-specific class of MADS box genes. A, Diagrammatic representation of the syntentic region in rice and B. distachyon that contains the ODDSOC-like genes (OsMADS51, BdOS1, and BdOS2), and the corresponding barley Unigene numbers and map locations. Arrows indicate the direction of transcription. B, Phylogenetic relationships between the ODDSOC-like genes of rice (OsMADS51), maize (ZmOS-like), sorghum (SbOS-like), barley (HvOS1 and HvOS2), wheat (TaAGL33, TaAGL41, and TaAGL42), and B. distachyon (BdOS1-like and BdOS2-like) based on a sequence alignment of the coding sequence for each gene.

Figure 2.

Vernalization-induced changes in HvOS1 and HvOS2 transcript levels. A, Expression of HvOS1 and HvOS2 in barley (cv Sonja) seedlings germinated in darkness at 20°C (NV, nonvernalized, white bars; n = 4) versus seedlings germinated in darkness at 4°C for 49 d (V, vernalized, black bars; n = 3) harvested at an equivalent stage of development. B, Expression levels of HvOS2 in fully expanded second leaves of nonvernalized plants (white bar; n = 3) versus vernalized plants (PV, postvernalization, black bar; n = 3) harvested in long days at the three-leaf stage, 10 d after the end of vernalization. C, Expression levels of HvOS2 in shoot apices (30–50 individual apices) from nonvernalized plants (0) or plants vernalized for 2, 5, or 9 weeks (w) and then grown in long days (n = 2). Plants were harvested at the three-leaf stage. All expression levels were assayed by qRT-PCR and are shown relative to ACTIN. Error bars show se (A and B) or range (C). Asterisks indicate P values of ANOVA: * P < 0.05, *** P < 0.001.

Figure 3.

Analysis of histone modifications at HvOS2 during vernalization. A, Relative abundance of H3K27me3 at the start of transcription for HvOS2 in nonvernalized plants (NV, black bar) and vernalized plants (PV, postvernalization, white bar; cv Sonja). B, Relative abundance of H3K4me3 at the start of transcription for HvOS2 in nonvernalized plants (black bar) and vernalized plants (white bar; cv Sonja). Error bars show sd.

Figure 4.

Expression of OS2 in different genotypes of wheat and barley. A, HvOS2 expression levels in nonvernalized (−) versus vernalized (+) plants (2 weeks old, grown in long days) from different barley cultivars, including three spring barleys that flower without vernalization, Morgenrot (lanes 1 and 2), Randolph (lanes 3 and 4), and Malta (lanes 5 and 6), and three vernalization-responsive winter barleys, Sonja (lanes 7 and 8), Hudson (lanes 9 and 10), and Mirra (lanes 11 and 12). Expression was assayed by high-stringency hybridization of RNA gel blots with a HvOS2-specific riboprobe. Ethidium bromide staining of ribosomal RNA is shown as a loading comparison. B, HvOS2 expression levels assayed by qRT-PCR in RNA from barley seedlings of a doubled haploid barley population (Sloop × Halcyon). Expression of HvOS2 was assayed in individual lines, relative to ACTIN, and the average expression levels of the different HvVRN1 genotypic classes were compared (VRN1, n = 22; VRN1-1, n = 19). C, Relative expression levels of TmOS2-like (TmAGL33) in the TmVRN1 deletion mutant (ΔVRN1; white bars) versus the wild-type parent strain (black bars). Expression was assayed in vernalized (V; n = 4) and nonvernalized (NV; n = 4) seedlings and in the leaves of plants grown for 1 week or 3 weeks (wild type [WT], n = 3; ΔVRN1, n = 2) in short days (SD) postvernalization (PV). Expression is shown relative to ACTIN. D, Relative expression levels of TmVRN1 in the conditions described in C. Error bars show se. Asterisks indicate P values of ANOVA: *** P < 0.001. ND, Not detected.

Figure 5.

Phenotypes of transgenic plants that overexpress HvOS2. A, Average days to head emergence (heading date) of transgenic barley lines overexpressing HvOS2 (black bars) versus sibling null segregant controls (plants from the same transgenic line that did not inherit the transgene; wild type [WT], white bars). B, Transgenic barley plants overexpressing HvOS2 versus null segregants at the fourth leaf stage (top), and apex images from the same stage (bottom). DR, Double ridges, the first sign of floral development; L, leaf primordia. C, Average length of the first and third leaves of plants overexpressing HvOxO2 (OxHvOS2-20; black bars) versus the null controls (white bars) at the sixth leaf stage (wild type, n = 5; OxHvOS2-20, n = 5). D, Average length of bulliform cells on the adaxial surface of mature leaves (first leaves [L1] and second leaves [L2]) from plants overexpressing HvOxO2 (OxHvOS2-20; black bars) versus wild-type siblings (white bars). L1, Wild type, n = 367 (cells) and OxHvOS2-20, n = 467; L2, wild type, n = 344 and OxHvOS2-20, n = 478. E, Scanning electron microscopy images of epidermal cells from the adaxial surface of mature leaves (first and second leaves). F, Average head and internode lengths of the primary tiller of plants overexpressing HvOxO2 (black bars) versus null segregants (white bars). P1, Peduncular internode; P-1, below P1; P-2, below P-1; P-3, below P-2. n = 15. Error bars show se. Asterisks indicate P values of ANOVA: * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 6.

Influence of vernalization on the expression of FPF1-like genes in short or long days. Expression of HvFPF1-like1 (HU14G14r; A) and HvFPF1-like2 (contig 18182; B) in the fully expanded second leaf (harvested at the third leaf stage), nonvernalized (white bars) versus vernalized plants (black bars), grown in long days (LD) or short days (SD). Expression was assayed by qRT-PCR and is shown relative to ACTIN. Error bars show se. Asterisks indicate P values of ANOVA: * P < 0.05 (minimum of three biological repeats).

Figure 7.

An extended model of the molecular genetic network that controls vernalization-induced flowering in temperate cereals. Low temperatures (cold) can transiently down-regulate OS2. Prolonged cold (vernalization) causes stable activation of VRN1. After vernalization, VRN1 down-regulates OS2, directly or indirectly. Consequently, FPF1s are derepressed. VRN1 also down-regulates VRN2 and allows activation of FT1 by long days (Trevaskis et al., 2007a; Distelfeld et al., 2009). Expression of FPF1s is induced as FT1 activity increases, and this promotes the transition to reproductive development at the shoot apex and cell elongation in the stem.