The molecular mechanisms of vernalization induced flowering in temperate cereals

Plants coordinate development with environmental cues to ensure flowering occurs under optimal seasonal conditions. Many plants from temperate regions only flower after exposure to prolonged cold: vernalization. While the molecular mechanisms of the vernalization response have been studied extensively in the model plant Arabidopsis, this seasonal flowering response has probably evolved independently in other plants. For instance, no homologues of FLOWER LOCUS C (FLC), the gene central to the vernalization response in Arabidopsis, have been found in economically important crops such as barley and wheat. Instead VERNALIZATION1 (VRN1) is central to the vernalization response in these plants. This highlights a need to study the vernalization response directly in cereal crop species. Previous studies have identified VRN1 as a master regulator of the vernalization response in cereals, but the extent to which other genes contribute is unclear. To identify genes that are potentially involved in regulating the vernalization response the Barley1 Affymetrix chip was used to compare gene expression in barley seedlings during short or prolonged cold treatments. Additionally, gene expression was assayed when plants were shifted to normal growth temperatures following prolonged cold treatment. This identified genes that show lasting changes in transcriptional activity, which might contribute to vernalization-induced flowering. Only a small group of genes showed a lasting change in activity when plants were shifted to warm conditions following prolonged cold. These included VRN1 and another MADS box gene, ODDSOC2 (OS2). OS2 belongs to a group of MADS box genes only found in grasses. Expression analysis showed that OS2 is down-regulated by cold and that long term repression of OS2 is dependent on VRN1. Overexpression of OS2 in barley delayed flowering and caused dwarfing. These phenotypes could be due to the down-regulation of FLOWERING PROMOTING FACTOR1 (FPF1)-like genes. In Arabidopsis FPF1 promotes flowering and elongation. Thus down-regulation of OS2 likely contributes to the acceleration of flowering through the de-repression of FPF1-like genes as daylength increases in spring. To determine if any components of the vernalization response pathway are conserved between Arabidopsis and temperate cereals, barley homologues of the Arabidopsis MADS box gene SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) were identified and characterised. In Arabidopsis SOC1 promotes flowering and is positively regulated by vernalization, long-days, and development. Similar to SOC1, the expression of a barley SOC1-like genes increases during development. Unlike Arabidopsis, the expression of a barley SOC1-like gene, HvSOC1-like1 was similar regardless of daylength suggesting that photoperiod does not regulate the expression of these genes. The regulation of HvSOC1-like genes by vernalization is also different. The expression of HvSOC1-like genes was lower in the leaves and crown tissue of vernalized plants versus non-vernalized plants. Over-expression of one of the barley SOC1-like genes, HvSOC1-like1, delayed flowering and caused dwarfing in barley plants. The phenotypes of these plants are different SOC1-like genes. Overall these data suggest that cereal SOC1-like genes have evolved differently and have different regulatory functions than SOC1 from Arabidopsis. Overall the findings from this thesis extend our understanding of the vernalization response in temperate cereals and highlight the difference of how this seasonal flowering response is regulated in temperate cereals and Arabidopsis. Since VRN1 is one of only a limited number of genes that initiates flowering in temperate cereals, understanding how VRN1 is regulated and identifying the targets of VRN1 will be of critical importance to understanding the mechanisms of seasonal flowering responses in these plants

ODDSOC2 Is a MADS Box Floral Repressor That Is Down-Regulated by Vernalization in Temperate Cereals

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

The influence of vernalization and daylength on expression of flowering-time genes in the shoot apex and leaves of barley (Hordeum vulgare).

Responses to prolonged low-temperature treatment of imbibed seeds (vernalization) were examined in barley (Hordeum vulgare). These occurred in two phases: the perception of prolonged cold, which occurred gradually at low temperatures, and the acceleration of reproductive development, which occurred after vernalization. Expression of the VERNALIZATION1 gene (HvVRN1) increased gradually in germinating seedlings during vernalization, both at the shoot apex and in the developing leaves. This occurred in darkness, independently of VERNALIZATION2 (HvVRN2), consistent with the hypothesis that expression of HvVRN1 is induced by prolonged cold independently of daylength flowering-response pathways. After vernalization, expression of HvVRN1 was maintained in the shoot apex and leaves. This was associated with accelerated inflorescence initiation and with down-regulation of HvVRN2 in the leaves. The largest determinant of HvVRN1 expression levels in vernalized plants was the length of seed vernalization treatment. Daylength did not influence HvVRN1 expression levels in shoot apices and typically did not affect expression in leaves. In the leaves of plants that had experienced a saturating seed vernalization treatment, expression of HvVRN1 was higher in long days, however. HvFT1 was expressed in the leaves of these plants in long days, which might account for the elevated HvVRN1 expression. Long-day up-regulation of HvVRN1 was not required for inflorescence initiation, but might accelerate subsequent stages of inflorescence development. Similar responses to seed vernalization were also observed in wheat (Triticum aestivum). These data support the hypothesis that VRN1 is induced by cold during winter to promote spring flowering in vernalization-responsive cereals

The Promoter of the Cereal VERNALIZATION1 Gene Is Sufficient for Transcriptional Induction by Prolonged Cold

The VERNALIZATION1 (VRN1) gene of temperate cereals is transcriptionally activated by prolonged cold during winter (vernalization) to promote flowering. To investigate the mechanisms controlling induction of VRN1 by prolonged cold, different regions of the VRN1 gene were fused to the GREEN FLUORESCENT PROTEIN (GFP) reporter and expression of the resulting gene constructs was assayed in transgenic barley (Hordeum vulgare). A 2 kb segment of the promoter of VRN1 was sufficient for GFP expression in the leaves and shoot apex of transgenic barley plants. Fluorescence increased at the shoot apex prior to inflorescence initiation and was subsequently maintained in the developing inflorescence. The promoter was also sufficient for low-temperature induction of GFP expression. A naturally occurring insertion in the proximal promoter, which is associated with elevated VRN1 expression and early flowering in some spring wheats, did not abolish induction of VRN1 transcription by prolonged cold, however. A translational fusion of the promoter and transcribed regions of VRN1 to GFP, VRN1::GFP, was localised to nuclei of cells at the shoot apex of transgenic barley plants. The distribution of VRN1::GFP at the shoot apex was similar to the expression pattern of the VRN1 promoter-GFP reporter gene. Fluorescence from the VRN1::GFP fusion protein increased in the developing leaves after prolonged cold treatment. These observations suggest that the promoter of VRN1 is targeted by mechanisms that trigger vernalization-induced flowering in economically important temperate cereal crops

Transcriptome Analysis of the Vernalization Response in Barley (Hordeum vulgare) Seedlings

Temperate cereals, such as wheat (Triticum spp.) and barley (Hordeum vulgare), respond to prolonged cold by becoming more tolerant of freezing (cold acclimation) and by becoming competent to flower (vernalization). These responses occur concomitantly during winter, but vernalization continues to influence development during spring. Previous studies identified VERNALIZATION1 (VRN1) as a master regulator of the vernalization response in cereals. The extent to which other genes contribute to this process is unclear. In this study the Barley1 Affymetrix chip was used to assay gene expression in barley seedlings during short or prolonged cold treatment. Gene expression was also assayed in the leaves of plants after prolonged cold treatment, in order to identify genes that show lasting responses to prolonged cold, which might contribute to vernalization-induced flowering. Many genes showed altered expression in response to short or prolonged cold treatment, but these responses differed markedly. A limited number of genes showed lasting responses to prolonged cold treatment. These include genes known to be regulated by vernalization, such as VRN1 and ODDSOC2, and also contigs encoding a calcium binding protein, 23-KD jasmonate induced proteins, an RNase S-like protein, a PR17d secretory protein and a serine acetyltransferase. Some contigs that were up-regulated by short term cold also showed lasting changes in expression after prolonged cold treatment. These include COLD REGULATED 14B (COR14B) and the barley homologue of WHEAT COLD SPECIFIC 19 (WSC19), which were expressed at elevated levels after prolonged cold. Conversely, two C-REPEAT BINDING FACTOR (CBF) genes showed reduced expression after prolonged cold. Overall, these data show that a limited number of barley genes exhibit lasting changes in expression after prolonged cold treatment, highlighting the central role of VRN1 in the vernalization response in cereals