Rie Kikuchi

Molecular and Functional Characterization of PEBP Genes in Barley Reveal the Diversification of Their Roles in Flowering

Five barley (Hordeum vulgare) PEBP (for phosphatidylethanolamine-binding protein) genes were analyzed to clarify their functional roles in flowering using transgenic, expression, and quantitative trait locus analyses. Introduction of HvTFL1 and HvMFT1 into rice (Oryza sativa) plants did not result in any changes in flowering, suggesting that these two genes have functions distinct from flowering. Overexpression of HvFT1, HvFT2, and HvFT3 in rice resulted in early heading, indicating that these FT-like genes can act as promoters of the floral transition. HvFT1 transgenic plants showed the most robust flowering initiation. In barley, HvFT1 was expressed at the time of shoot meristem phase transition. These results suggest that HvFT1 is the key gene responsible for flowering in the barley FT-like gene family. HvFT2 transgenic plants also showed robust flowering initiation, but HvFT2 was expressed only under short-day (SD) conditions during the phase transition, suggesting that its role is limited to specific photoperiodic conditions in barley. Flowering activity in HvFT3 transgenic rice was not as strong and was modulated by the photoperiod. These results suggest that HvFT3 functions in flowering promotion but that its effect is indirect. HvFT3 expression was observed in Morex, a barley cultivar carrying a dominant allele of Ppd-H2, a major quantitative trait locus for flowering under SD conditions, although no expression was detected in Steptoe, a cultivar carrying ppd-H2. HvFT3 was expressed in Morex under both long-day and SD conditions, although its expression was increased under SD conditions. HvFT3 was mapped to chromosome 1HL, the same chromosome that carries Ppd-H2. Genomic sequence analyses revealed that Morex possesses an intact HvFT3 gene, whereas most of this gene has been lost in Steptoe. These data strongly suggest that HvFT3 may be identical to Ppd-H2.

A genetic network of flowering‐time genes in wheat leaves, in which an APETALA1/FRUITFULL‐like gene, VRN1, is upstream of FLOWERING LOCUS T

To elucidate the genetic mechanism of flowering in wheat, we performed expression, mutant and transgenic studies of flowering-time genes. A diurnal expression analysis revealed that a flowering activator VRN1, an APETALA1/FRUITFULL homolog in wheat, was expressed in a rhythmic manner in leaves under both long-day (LD) and short-day (SD) conditions. Under LD conditions, the upregulation of VRN1 during the light period was followed by the accumulation of FLOWERING LOCUS T (FT) transcripts. Furthermore, FT was not expressed in a maintained vegetative phase (mvp) mutant of einkorn wheat (Triticum monococcum), which has null alleles of VRN1, and never transits from the vegetative to the reproductive phase. These results suggest that VRN1 is upstream of FT and upregulates the FT expression under LD conditions. The overexpression of FT in a transgenic bread wheat (Triticum aestivum) caused extremely early heading with the upregulation of VRN1 and the downregulation of VRN2, a putative repressor gene of VRN1. These results suggest that in the transgenic plant, FT suppresses VRN2 expression, leading to an increase in VRN1 expression. Based on these results, we present a model for a genetic network of flowering-time genes in wheat leaves, in which VRN1 is upstream of FT with a positive feedback loop through VRN2. The mvp mutant has a null allele of VRN2, as well as of VRN1, because it was obtained from a spring einkorn wheat strain lacking VRN2. The fact that FT is not expressed in the mvp mutant supports the present model.

The differential expression of HvCO9, a member of the CONSTANS-like gene family, contributes to the control of flowering under short-day conditions in barley

HvCO9 was characterized to elucidate the barley flowering control mechanisms and to investigate the functional diversification of the barley CONSTANS-like (CO-like) genes in flowering. HvCO9 was located on the same chromosome, 1HL, as Ppd-H2 (HvFT3), which is a positive regulator of short-day (SD) flowering. A phylogenetic analysis showed that HvCO9 was located on the same branch of the CO-like gene tree as rice Ghd7 and the barley and wheat VRN2 genes, which are all negative regulators of flowering. High level HvCO9 expressions were observed under SD conditions, whereas its expression levels were quite low under long-day (LD) conditions. HvCO9 expression correlated with HvFT1 and HvFT2 expression under SD conditions, although no clear effect of HvCO9 on HvFT3 expression, or vice versa, under SD conditions was observed. The over-expression of HvCO9 in rice plants produced a remarkable delay in flowering. In transgenic rice, the expression levels of the flowering-related Ehd1 gene, which is a target gene of Ghd7, and its downstream genes were suppressed, causing a delay in flowering. These results suggest that HvCO9 may act as a negative regulator of flowering under non-inductive SD conditions in barley; this activity is similar to that of rice Ghd7 under non-inductive LD conditions, but the functional targets of these genes may be different. Our results indicate that barley has developed its own pathways to control flowering by using homologous genes with modifications for the timing of expression. Further, it is hypothesized that each pathway may target different genes after gene duplication or species diversification.

Variations for Fusarium head blight resistance associated with genomic diversity in different sources of the resistant wheat cultivar 'Sumai 3'

Fusarium head blight (FHB), caused by Fusarium graminearum, is a serious disease of wheat (Triticum aestivum L.) associated with contamination by the mycotoxin deoxynivalenol (DON). The FHB-resistant wheat cultivar ‘Sumai 3’ has been used extensively around the world. The existence of variation in FHB resistance among ‘Sumai 3’ accessions has been discussed. In this study, genetic variation among ‘Sumai 3’ accessions collected from six countries were identified using SSR markers; our results demonstrate unique chromosome regions in Sumai 3-AUT and Sumai 3-JPN (‘Sumai 3’ accessions from Austria and Japan, respectively). Field evaluation indicated strong resistance to FHB in Sumai 3-AUT. The polymorphic rate (number of polymorphic markers/number of available markers × 100) based on a DArT array was 12.5% between the two ‘Sumai 3’ accessions. Genotyping for DNA markers flanking FHB-resistant quantitative trait loci (QTLs) revealed genetic variations for the QTL regions on 5AS and 2DS; however, no variation was observed for the QTL regions on 3BS and 6B. Thus, the variation in FHB resistance among ‘Sumai 3’ accessions in the field is due to genetic diversity.

PnMADS1, encoding an StMADS11-clade protein, acts as a repressor of flowering in Pharbitis nil.

We previously isolated PnMADS1, a MADS-box transcription factor and member of the functionally diverse StMADS11 clade of the MADS-box family, from Pharbitis nil, which is a typical SD plant. However, its precise function remained unclear. To investigate the biological role of PnMADS1, and especially its involvement in flowering, we constructed transgenic P. nil plants that overexpresses or underexpresses PnMADS1. PnMADS1-RNAi transformants had an increased number of flower buds, whereas overexpression of PnMADS1 led to a decrease in the number of flower buds, although both transgenic plants maintained the photoperiodic responses of flowering. These results suggest that PnMADS1 negatively regulates floral evocation from the vegetative phase to the reproductive phase but it has no essential role in floral induction by photoperiodic signals. Results of yeast two-hybrid experiments revealed that PnMADS1 can interact with itself, suggesting that this protein functions in floral evocation as a homodimer. PnMADS1 also interacts with PnSAH3, an AP1-clade protein, suggesting that PnMADS1 has a functional role in flower formation as a heterodimer with other MADS-box protein(s).