W. James Peacock

The FLF MADS box gene: a repressor of flowering in Arabidopsis regulated by vernalization and methylation.

A MADS box gene, FLF (for FLOWERING LOCUS F), isolated from a late-flowering, T-DNA–tagged Arabidopsis mutant, is a semidominant gene encoding a repressor of flowering. The FLF gene appears to integrate the vernalization-dependent and autonomous flowering pathways because its expression is regulated by genes in both pathways. The level of FLF mRNA is downregulated by vernalization and by a decrease in genomic DNA methylation, which is consistent with our previous suggestion that vernalization acts to induce flowering through changes in gene activity that are mediated through a reduction in DNA methylation. The flf-1 mutant requires a greater than normal amount of an exogenous gibberellin (GA3) to decrease flowering time compared with the wild type or with vernalization-responsive late-flowering mutants, suggesting that the FLF gene product may block the promotion of flowering by GAs. FLF maps to a region on chromosome 5 near the FLOWERING LOCUS C gene, which is a semidominant repressor of flowering in late-flowering ecotypes of Arabidopsis.

Expression Profile Analysis of the Low-Oxygen Response in Arabidopsis Root Cultures

We used DNA microarray technology to identify genes involved in the low-oxygen response of Arabidopsis root cultures. A microarray containing 3500 cDNA clones was screened with cDNA samples taken at various times (0.5, 2, 4, and 20 h) after transfer to low-oxygen conditions. A package of statistical tools identified 210 differentially expressed genes over the four time points. Principal component analysis showed the 0.5-h response to contain a substantially different set of genes from those regulated differentially at the other three time points. The differentially expressed genes included the known anaerobic proteins as well as transcription factors, signal transduction components, and genes that encode enzymes of pathways not known previously to be involved in low-oxygen metabolism. We found that the regulatory regions of genes with a similar expression profile contained similar sequence motifs, suggesting the coordinated transcriptional control of groups of genes by common sets of regulatory factors.

MADS box genes control vernalization-induced flowering in cereals

By comparing expression levels of MADS box transcription factor genes between near-isogenic winter and spring lines of bread wheat, Triticum aestivum, we have identified WAP1 as the probable candidate for the Vrn-1 gene, the major locus controlling the vernalization flowering response in wheat. WAP1 is strongly expressed in spring wheats and moderately expressed in semispring wheats, but is not expressed in winter wheat plants that have not been exposed to vernalization treatment. Vernalization promotes flowering in winter wheats and strongly induces expression of WAP1. WAP1 is located on chromosome 5 in wheat and, by synteny with other cereal genomes, is likely to be collocated with Vrn-1. These results in hexaploid bread wheat cultivars extend the conclusion made by Yan et al. [Yan, L., Loukoianov, A., Tranquilli, G., Helguera, M., Fahima, T. & Dubcovsky, J. (2003) Proc. Natl. Acad. Sci. USA 100, 6263–6268] in the diploid wheat progenitor Triticum monococcum that WAP1 (TmAP1) corresponds to the Vrn-1 gene. The barley homologue of WAP1, BM5, shows a similar pattern of expression to WAP1 and TmAP1. BM5 is not expressed in winter barleys that have not been vernalized, but as with WAP1, expression of BM5 is strongly induced by vernalization treatment. In spring barleys, the level of BM5 expression is determined by interactions between the Vrn-H1 locus and a second locus for spring habit, Vrn-H2. There is now evidence that AP1-like genes determine the time of flowering in a range of cereal and grass species.

The Arabidopsis thaliana vernalization response requires a polycomb-like protein complex that also includes VERNALIZATION INSENSITIVE 3

In Arabidopsis thaliana, the promotion of flowering by cold temperatures, vernalization, is regulated via a floral-repressive MADS box transcription factor, FLOWERING LOCUS C (FLC). Vernalization leads to the epigenetic repression of FLC expression, a process that requires the polycomb group (PcG) protein VERNALIZATION 2 (VRN2) and the plant homeodomain protein VERNALIZATION INSENSITIVE 3 (VIN3). We demonstrate that the repression of FLC by vernalization requires homologues of other Polycomb Repressive Complex 2 proteins and VRN2. We show in planta that VRN2 and VIN3 are part of a large protein complex that can include the PcG proteins FERTILIZATION INDEPENDENT ENDOSPERM, CURLY LEAF, and SWINGER. These findings suggest a single protein complex is responsible for histone deacetylation at FLC and histone methylation at FLC in vernalized plants. The abundance of the complex increases during vernalization and declines after plants are returned to higher temperatures, consistent with the complex having a role in establishing FLC repression.

DNA methylation, a key regulator of plant development and other processes

Recent research has demonstrated that DNA methylation plays an integral role in regulating the timing of flowering and in endosperm development. The identification of key genes controlling these processes, the expression of which is altered in plants with low methylation, opens the way to understanding how DNA methylation regulates plant development.

Different Regulatory Regions Are Required for the Vernalization-Induced Repression of FLOWERING LOCUS C and for the Epigenetic Maintenance of Repression

Vernalization, the promotion of flowering by a prolonged period of low temperature, results in repression of the floral repressor FLOWERING LOCUS C (FLC) and in early flowering. This repression bears the hallmark of an epigenetic event: the low expression state is maintained over many cell division cycles, but expression is derepressed in progeny. We show that the two stages of the response of FLC to vernalization, the repression of FLC and the maintenance of the repression during growth at normal temperatures after vernalization, are mediated through different regions of the FLC gene. Both promoter and intragenic regions are required for the responses. We also identify a 75-bp region in the FLC promoter that, in addition to intragenic sequences, is required for expression in nonvernalized plants.

The molecular biology of seasonal flowering-responses in Arabidopsis and the cereals

BACKGROUND In arabidopsis (Arabidopsis thaliana), FLOWERING LOCUS T (FT) and FLOWERING LOCUS C (FLC) play key roles in regulating seasonal flowering-responses to synchronize flowering with optimal conditions. FT is a promoter of flowering activated by long days and by warm conditions. FLC represses FT to delay flowering until plants experience winter. SCOPE The identification of genes controlling flowering in cereals allows comparison of the molecular pathways controlling seasonal flowering-responses in cereals with those of arabidopsis. The role of FT has been conserved between arabidopsis and cereals; FT-like genes trigger flowering in response to short days in rice or long days in temperate cereals, such as wheat (Triticum aestivum) and barley (Hordeum vulgare). Many varieties of wheat and barley require vernalization to flower but FLC-like genes have not been identified in cereals. Instead, VERNALIZATION2 (VRN2) inhibits long-day induction of FT-like1 (FT1) prior to winter. VERNALIZATION1 (VRN1) is activated by low-temperatures during winter to repress VRN2 and to allow the long-day response to occur in spring. In rice (Oryza sativa) a VRN2-like gene Ghd7, which influences grain number, plant height and heading date, represses the FT-like gene Heading date 3a (Hd3a) in long days, suggesting a broader role for VRN2-like genes in regulating day-length responses in cereals. Other genes, including Early heading date (Ehd1), Oryza sativa MADS51 (OsMADS51) and INDETERMINATE1 (OsID1) up-regulate Hd3a in short days. These genes might account for the different day-length response of rice compared with the temperate cereals. No genes homologous to VRN2, Ehd1, Ehd2 or OsMADS51 occur in arabidopsis. CONCLUSIONS It seems that different genes regulate FT orthologues to elicit seasonal flowering-responses in arabidopsis and the cereals. This highlights the need for more detailed study into the molecular basis of seasonal flowering-responses in cereal crops or in closely related model plants such as Brachypodium distachyon.

HvVRN2 Responds to Daylength, whereas HvVRN1 Is Regulated by Vernalization and Developmental Status

Two genetic loci control the vernalization response in winter cereals; VRN1, which encodes an AP1-like MADS-box transcription factor, and VRN2, which has been mapped to a chromosome region containing ZCCT zinc finger transcription factor genes. We examined whether daylength regulates expression of HvVRN1 and HvVRN2. In a vernalization-responsive winter barley (Hordeum vulgare), expression of HvVRN1 is regulated by vernalization and by development, but not by daylength. Daylength affected HvVRN1 expression in only one of six vernalization-insensitive spring barleys examined and so cannot be a general feature of regulation of this gene. In contrast, daylength is the major determinant of expression levels of two ZCCT genes found at the barley VRN2 locus, HvZCCTa and HvZCCTb. In winter barley, high levels of HvZCCTa and HvZCCTb expression were detected only when plants were grown in long days. During vernalization in long-day conditions, HvVRN1 is induced and expression of HvZCCTb is repressed. During vernalization under short days, induction of HvVRN1 occurs without changes in HvZCCTa and HvZCCTb expression. Analysis of HvZCCTa and HvZCCTb expression levels in a doubled haploid population segregating for different vernalization and daylength requirements showed that HvVRN1 genotype determines HvZCCTa and HvZCCTb expression levels. We conclude that the vernalization response is mediated through HvVRN1, whereas HvZCCTa and HvZCCTb respond to daylength cues to repress flowering under long days in nonvernalized plants.

Low-Temperature and Daylength Cues Are Integrated to Regulate FLOWERING LOCUS T in Barley

Interactions between flowering time genes were examined in a doubled haploid barley (Hordeum vulgare) population segregating for H. vulgare VERNALIZATION1 (HvVRN1), HvVRN2, and PHOTOPERIOD1 (PPD-H1). A deletion allele of HvVRN2 was associated with rapid inflorescence initiation and early flowering, but only in lines with an active allele of PPD-H1. In these lines, the floral promoter FLOWERING LOCUS T (HvFT1) was expressed at high levels without vernalization, and this preceded induction of HvVRN1. Lines with the deletion allele of HvVRN2 and the inactive ppd-H1 allele did not undergo rapid inflorescence initiation and were late flowering. These data suggest that HvVRN2 counteracts PPD-H1 to prevent flowering prior to vernalization. An allele of HvVRN1 that is expressed at high basal levels (HvVRN1-1) was associated with rapid inflorescence initiation regardless of HvVRN2 or PPD-H1 genotype. HvFT1 was expressed without vernalization in lines with the HvVRN1-1 allele and HvFT1 transcript levels were highest in lines with the active PPD-H1 allele; this correlated with rapid apex development postinflorescence initiation. Thus, expression of HvVRN1 promotes inflorescence initiation and up-regulates HvFT1. Analysis of HvVRN1 expression in different genetic backgrounds postvernalization showed that HvVRN2, HvFT1, and PPD-H1 are unlikely to play a role in low-temperature induction of HvVRN1. In a vernalization responsive barley, HvFT1 is not induced by low temperatures alone, but can be induced by long days following prolonged low-temperature treatment. We conclude that low-temperature and daylength flowering-response pathways are integrated to control expression of HvFT1 in barley, and that this might occur through regulation of HvVRN2 activity.

Resetting of FLOWERING LOCUS C expression after epigenetic repression by vernalization

The epigenetic repression of FLOWERING LOCUS C (FLC) in winter-annual ecotypes of Arabidopsis by prolonged cold ensures that plants flower in spring and not during winter. Resetting of the FLC expression level in progeny is an important step in the life cycle of the plant. We show that both the paternally derived and the maternally derived FLC:GUS genes are reset to activity but that the timing of their first expression differs. The paternal FLC:GUS gene in vernalized plants is expressed in the male reproductive organs, the anthers, in both somatic tissue and in the sporogenous pollen mother cells, but there is no expression in mature pollen. In the progeny generation, the paternally derived FLC:GUS gene is expressed in the single-celled zygote (fertilized egg cell) and through embryo development, but not in the fertilized central cell, which generates the endosperm of the progeny seed. FLC:GUS is not expressed during female gametogenesis, with the maternally derived FLC:GUS being first expressed in the early multicellular embryo. We show that FLC activity during late embryo development is a prerequisite for the repressive action of FLC on flowering.