Ben Trevaskis

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

BACKGROUNDnIn 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.nnnSCOPEnThe 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.nnnCONCLUSIONSnIt 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.

Vernalization-induced flowering in cereals is associated with changes in histone methylation at the VERNALIZATION1 gene

Prolonged exposure to low temperatures (vernalization) accelerates the transition to reproductive growth in many plant species, including the model plant Arabidopsis thaliana and the economically important cereal crops, wheat and barley. Vernalization-induced flowering is an epigenetic phenomenon. In Arabidopsis, stable down-regulation of FLOWERING LOCUS C (FLC) by vernalization is associated with changes in histone modifications at FLC chromatin. In cereals, the vernalization response is mediated by stable induction of the floral promoter VERNALIZATION1 (VRN1), which initiates reproductive development at the shoot apex. We show that in barley (Hordeum vulgare), repression of HvVRN1 before vernalization is associated with high levels of histone 3 lysine 27 trimethylation (H3K27me3) at HvVRN1 chromatin. Vernalization caused increased levels of histone 3 lysine 4 trimethylation (H3K4me3) and a loss of H3K27me3 at HvVRN1, suggesting that vernalization promotes an active chromatin state at VRN1. Levels of these histone modifications at 2 other flowering-time genes, VERNALIZATION2 and FLOWERING LOCUS T, were not altered by vernalization. Our study suggests that maintenance of an active chromatin state at VRN1 is likely to be the basis for epigenetic memory of vernalization in cereals. Thus, regulation of chromatin state is a feature of epigenetic memory of vernalization in Arabidopsis and the cereals; however, whereas vernalization-induced flowering in Arabidopsis is mediated by epigenetic regulation of the floral repressor FLC, this phenomenon in cereals is mediated by epigenetic regulation of the floral activator, VRN1.

Increased level of hemoglobin 1 enhances survival of hypoxic stress and promotes early growth in Arabidopsis thaliana

Overexpression of a class 1 Hb (GLB1) protects Arabidopsis thaliana plants from the effects of severe hypoxia. Overexpression of the bifunctional symbiotic Hb (GLB1S) from Parasponia andersonii in A. thaliana also increases survival after hypoxia. Plants overexpressing the Hb 1 protein, mutated to have a low oxygen affinity, are as susceptible to hypoxia as WT plants, suggesting that the protection against hypoxia depends on the ability of the Hb to bind ligands, such as oxygen, with high affinity. A mild hypoxia pretreatment (5%) induces the Hb gene and increases the survival of plants after severe hypoxic treatment (0.1%). These results with Hb 1 show that plant Hbs have a role other than in nitrogen-fixing root nodules. Plants overexpressing the GLB1 protein show early vigorous growth in nonhypoxic conditions and are 50% larger in weight than the controls at 14 days. The constitutive expression of GLB1 also resulted in a reduced number of root hairs and increased number of laterals in the root system.

Expression and evolution of functionally distinct haemoglobin genes in plants

Haemoglobin genes have been found in a number of plant species, but the number of genes known has been too small to allow effective evolutionary inferences. We present nine new non-symbiotic haemoglobin sequences from a range of plants, including classxa01 haemoglobins from cotton, Citrus and tomato, classxa02 haemoglobins from cotton, tomato, sugar beet and canola and two haemoglobins from the non-vascular plants, Marchantia polymorpha (a liverwort) and Physcomitrella patens (a moss). Our molecular phylogenetic analysis of all currently known non-symbiotic haemoglobin genes and a selection of symbiotic haemoglobins have confirmed the existence of two distinct classes of haemoglobin genes in the dicots. It is likely that all dicots have both classxa01 and classxa02 non-symbiotic haemoglobin genes whereas in monocots we have detected only classxa01 genes. The symbiotic haemoglobins from legumes and Casuarina are related to the classxa02 non-symbiotic haemoglobins, whilst the symbiotic haemoglobin from Parasponia groups with the classxa01 non-symbiotic genes. Probably, there have been two independent recruitments of symbiotic haemoglobins. Although the functions of the two non-symbiotic haemoglobins remain unknown, their patterns of expression within plants suggest different functions. We examined the expression in transgenic plants of the two non-symbiotic haemoglobins from Arabidopsis using promoter fusions to a GUS reporter gene. The Arabidopsis GLB1 and GLB2 genes are likely to be functionally distinct. The classxa02 haemoglobin gene (GLB2) is expressed in the roots, leaves and inflorescence and can be induced in young plants by cytokinin treatment in contrast to the classxa01 gene (GLB1) which is active in germinating seedlings and can be induced by hypoxia and increased sucrose supply, but not by cytokinin treatment.

Identification with Proteomics of Novel Proteins Associated with the Peribacteroid Membrane of Soybean Root Nodules

Soybean peribacteroid membrane (PBM) proteins were isolated from nitrogen-fixing root nodules and subjected to N-terminal sequencing. Sequence data from 17 putative PBM proteins were obtained. Six of these proteins are homologous to proteins of known function. These include three chaperones (HSP60, BiP [HSP70], and PDI) and two proteases (a serine and a thiol protease), all of which are involved in some aspect of protein processing in plants. The PBM homologs of these proteins may play roles in protein translocation, folding, maturation, or degradation in symbiosomes. Two proteins are homologous to known, nodule-specific proteins from soybean, nodulin 53b and nodulin 26B. Although the function of these nodulins is unknown, nodulin 53b has independently been shown to be associated with the PBM. All of the eight proteins with identifiable homologs are likely to be peripheral rather than integral membrane proteins. Possible reasons for this apparent bias are discussed. The identification of homologs of HSP70 and HSP60 associated with the PBM is the first evidence that the molecular machinery for co- or post-translational import of cytoplasmic proteins is present in symbiosomes. This has important implications for the biogenesis of this unique, nitrogen-fixing organelle.

GmZIP1 Encodes a Symbiosis-specific Zinc Transporter in Soybean

The importance of zinc in organisms is clearly established, and mechanisms involved in zinc acquisition by plants have recently received increased interest. In this report, the identification, characterization and location of GmZIP1, the first soybean member of the ZIP family of metal transporters, are described. GmZIP1 was found to possess eight putative transmembrane domains together with a histidine-rich extra-membrane loop. By functional complementation of zrt1zrt2 yeast cells no longer able to take up zinc, GmZIP1 was found to be highly selective for zinc, with an estimated K m value of 13.8 μm. Cadmium was the only other metal tested able to inhibit zinc uptake in yeast. An antibody raised against GmZIP1 specifically localized the protein to the peribacteroid membrane, an endosymbiotic membrane in nodules resulting from the interaction of the plant with its microsymbiont. The specific expression of GmZIP1 in nodules was confirmed by Northern blot, with no expression in roots, stems, or leaves of nodulated soybean plants. Antibodies to GmZIP1 inhibited zinc uptake by symbiosomes, indicating that at least some of the zinc uptake observed in isolated symbiosomes could be attributed to GmZIP1. The orientation of the protein in the membrane and its possible role in the symbiosis are discussed.

Short Vegetative Phase-Like MADS-Box Genes Inhibit Floral Meristem Identity in Barley

Analysis of the functions of Short Vegetative Phase (SVP)-like MADS-box genes in barley (Hordeum vulgare) indicated a role in determining meristem identity. Three SVP-like genes are expressed in vegetative tissues of barley: Barley MADS1 (BM1), BM10, and Vegetative to Reproductive Transition gene 2. These genes are induced by cold but are repressed during floral development. Ectopic expression of BM1 inhibited spike development and caused floral reversion in barley, with florets at the base of the spike replaced by tillers. Head emergence was delayed in plants that ectopically express BM1, primarily by delayed development after the floral transition, but expression levels of the barley VRN1 gene (HvVRN1) were not affected. Ectopic expression of BM10 inhibited spike development and caused partial floral reversion, where florets at the base of the spike were replaced by inflorescence-like structures, but did not affect heading date. Floral reversion occurred more frequently when BM1 and BM10 ectopic expression lines were grown in short-day conditions. BM1 and BM10 also inhibited floral development and caused floral reversion when expressed in Arabidopsis (Arabidopsis thaliana). We conclude that SVP-like genes function to suppress floral meristem identity in winter cereals.