Genome-wide association analyses reveal complex genetic architecture underlying natural variation for flowering time in canola.

Optimum flowering time is the key to maximize canola production in order to meet global demand of vegetable oil, biodiesel and canola-meal. We reveal extensive variation in flowering time across diverse genotypes of canola under field, glasshouse and controlled environmental conditions. We conduct a genome-wide association study and identify 69 single nucleotide polymorphism (SNP) markers associated with flowering time, which are repeatedly detected across experiments. Several associated SNPs occur in clusters across the canola genome; seven of them were detected within 20 Kb regions of a priori candidate genes; FLOWERING LOCUS T, FRUITFUL, FLOWERING LOCUS C, CONSTANS, FRIGIDA, PHYTOCHROME B and an additional five SNPs were localized within 14 Kb of a previously identified quantitative trait loci for flowering time. Expression analyses showed that among FLC paralogs, BnFLC.A2 accounts for ~23% of natural variation in diverse accessions. Genome-wide association analysis for FLC expression levels mapped not only BnFLC.C2 but also other loci that contribute to variation in FLC expression. In addition to revealing the complex genetic architecture of flowering time variation, we demonstrate that the identified SNPs can be modelled to predict flowering time in diverse canola germplasm accurately and hence are suitable for genomic selection of adaptative traits in canola improvement programmes.

Epigenetic regulation of flowering.

The acceleration of flowering by prolonged low temperature treatment (vernalization) has unique properties including the floral transition occurring at a time separate from the vernalization treatment. This implies the vernalization condition is inherited through mitotic divisions, but this vernalized state is not inherited from one generation to the next. FLC, the key gene mediating this response in the Arabidopsis is repressed by histone modifications involving the VRN2 protein complex. Other protein complexes participate in activating the gene. While many plant species depend on vernalization for optimising flowering time, the genes involved differ between dicot and monocot plants in both Arabidopsis and cereals, vernalization regulates photoperiod control of flowering by preventing the induction of the floral promoter FT by long days in autumn but allowing induction of FT in spring and hence flowering occurs at an optimal time in the annual life cycle.

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 class 1 haemoglobins from cotton, Citrus and tomato, class 2 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 class 1 and class 2 non-symbiotic haemoglobin genes whereas in monocots we have detected only class 1 genes. The symbiotic haemoglobins from legumes and Casuarina are related to the class 2 non-symbiotic haemoglobins, whilst the symbiotic haemoglobin from Parasponia groups with the class 1 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 class 2 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 class 1 gene (GLB1) which is active in germinating seedlings and can be induced by hypoxia and increased sucrose supply, but not by cytokinin treatment.

GAMYB-like genes, flowering, and gibberellin signaling in Arabidopsis.

We have identified three Arabidopsis genes with GAMYB-like activity, AtMYB33, AtMYB65, and AtMYB101, which can substitute for barley (Hordeum vulgare) GAMYB in transactivating the barley alpha-amylase promoter. We have investigated the relationships between gibberellins (GAs), these GAMYB-like genes, and petiole elongation and flowering of Arabidopsis. Within 1 to 2 d of transferring plants from short- to long-day photoperiods, growth rate and erectness of petioles increased, and there were morphological changes at the shoot apex associated with the transition to flowering. These responses were accompanied by accumulation of GAs in the petioles (GA(1) by 11-fold and GA(4) by 3-fold), and an increase in expression of AtMYB33 at the shoot apex. Inhibition of GA biosynthesis using paclobutrazol blocked the petiole elongation induced by long days. Causality was suggested by the finding that, with GA treatment, plants flowered in short days, AtMYB33 expression increased at the shoot apex, and the petioles elongated and grew erect. That AtMYB33 may mediate a GA signaling role in flowering was supported by its ability to bind to a specific 8-bp sequence in the promoter of the floral meristem-identity gene, LEAFY, this same sequence being important in the GA response of the LEAFY promoter. One or more of these AtMYB genes may also play a role in the root tip during germination and, later, in stem tissue. These findings extend our earlier studies of GA signaling in the Gramineae to include a dicot species, Arabidopsis, and indicate that GAMYB-like genes may mediate GA signaling in growth and flowering responses.

Control of early seed development.

Seed development requires coordinated expression of embryo and endosperm and has contributions from both sporophytic and male and female gametophytic genes. Genetic and molecular analyses in recent years have started to illuminate how products of these multiple genes interact to initiate seed development. Imprinting or differential expression of paternal and maternal genes seems to be involved in controlling seed development, presumably by controlling gene expression in developing endosperm. Epigenetic processes such as chromatin remodeling and DNA methylation affect imprinting of key seed-specific genes; however, the identity of many of these genes remains unknown. The discovery of FIS genes has illuminated control of autonomous endosperm development, a component of apomixis, which is an important developmental and agronomic trait. FIS genes are targets of imprinting, and the genes they control in developing endosperm are also regulated by DNA methylation and chromatin remodeling genes. These results define some exciting future areas of research in seed development.

A plastid envelope location of Arabidopsis ent-kaurene oxidase links the plastid and endoplasmic reticulum steps of the gibberellin biosynthesis pathway.

We have used fusions of gibberellin biosynthesis enzymes to green fluorescent protein (GFP) to determine the subcellular localization of the early steps of the pathway. Gibberellin biosynthesis from geranylgeranyl diphosphate is catalysed by enzymes of the terpene cyclase, cytochrome P450 mono-oxygenase and 2-oxoglutarate-dependent dioxygenase classes. We show that the N-terminal pre-sequences of the Arabidopsis thaliana terpene cyclases copalyl diphosphate synthase (AtCPS1) and ent-kaurene synthase (AtKS1) direct GFP to chloroplasts in transient assays following microprojectile bombardment of tobacco leaves. The AtKS1-GFP fusion is also imported by isolated pea chloroplasts. The N-terminal portion of the cytochrome P450 protein ent-kaurene oxidase (AtKO1) directs GFP to chloroplasts in tobacco leaf transient assays. Chloroplast import assays with 35S-labelled AtKO1 protein show that it is targeted to the outer face of the chloroplast envelope. The leader sequences of the two ent-kaurenoic acid oxidases (AtKAO1 and AtKAO2) from Arabidopsis direct GFP to the endoplasmic reticulum. These data suggest that the AtKO1 protein links the plastid- and endoplasmic reticulum-located steps of the gibberellin biosynthesis pathway by association with the outer envelope of the plastid.

The Arabidopsis AMP1 gene encodes a putative glutamate carboxypeptidase.

Arabidopsis amp1 mutants show pleiotropic phenotypes, including altered shoot apical meristems, increased cell proliferation, polycotyly, constitutive photomorphogenesis, early flowering time, increased levels of endogenous cytokinin, and increased cyclin cycD3 expression. We have isolated the AMP1 gene by map-based cloning. The AMP1 cDNA encodes a 706;-amino acid polypeptide with significant similarity to glutamate carboxypeptidases. The AMP1 mRNA was expressed in all tissues examined, with higher expression in roots, stems, inflorescences, and siliques. Microarray analysis identified four mRNA species with altered expression in two alleles of amp1, including upregulation of CYP78A5, which has been shown to mark the shoot apical meristem boundary. The similarity of the AMP1 protein to glutamate carboxypeptidases, and in particular to N-acetyl alpha-linked acidic dipeptidases, suggests that the AMP1 gene product modulates the level of a small signaling molecule that acts to regulate a number of aspects of plant development, in particular the size of the apical meristem.

A hemoglobin from plants homologous to truncated hemoglobins of microorganisms.

We have identified a nuclear-encoded Hb from plants (GLB3) that has a central domain similar to the "truncated" Hbs of bacteria, protozoa, and algae. The three-dimensional structure of these Hbs is a 2-on-2 arrangement of alpha-helices, distinct from the 3-on-3 arrangement of the standard globin fold [Pesce, A., Couture, M., Dewilde, S., Guertin, M., Yamauchi, K., Ascenzi, P., Moens, L. & Bolognesi, M. (2000) EMBO J. 19, 2424-2434]. GLB3-like genes are not found in animals or yeast, but our analysis reveals that they are present in a wide range of Angiosperms and a Bryophyte. Although cyanobacteria and Chlamydomonas have 2-on-2 Hbs (GLBN), GLB3 is more likely related to GLBO-type 2-on-2 Hbs from bacteria. Consequently, GLB3 is unlikely to have arisen from a horizontal transfer between the chloroplast and nuclear genomes. Arabidopsis thaliana GLB3 protein exhibits unusual concentration-independent binding of O(2) and CO. The absorbance spectrum of deoxy-GLB3 is unique; the protein forms a transient six-coordinate structure after reduction and deoxygenation, which slowly converts to a five-coordinate structure. In A. thaliana, GLB3 is expressed throughout the plant but responds to none of the treatments that induce plant 3-on-3 Hbs. Our analysis of the sequence, ligand interactions, and expression profile of GLB3 indicates that this protein has unique biochemical properties, evolutionary history, and, most likely, a function distinct from those of other plant Hbs.

Site specificity of the Arabidopsis METI DNA methyltransferase demonstrated through hypermethylation of the superman locus.

Plants with low levels of DNA methylation show a range of developmental abnormalities including homeotic transformation of floral organs. Two independent DNA METHYLTRANSFERASEI (METI) antisense transformants with low levels of DNA methylation had flowers with increased numbers of stamens which resembled flowers seen on the loss-of-function superman (sup) mutant plants and on transgenic plants that ectopically express APETALA3 (AP3). These METI antisense plants have both increased and decreased methylation in and around the sup gene, compared with untransformed controls. DNA from the antisense plants was demethylated at least 4 kb upstream of the sup gene, while there was dense methylation around the start of transcription and within the coding region of this gene; these regions were unmethylated in control DNA. Methylation within the sup gene was correlated with an absence of SUP transcripts. The pattern and density of methylation was heterogeneous among different DNA molecules from the same plant, with some molecules being completely unmethylated. Methylcytosine occurred in asymmetric sites and in symmetric CpA/TpG but rarely in CpG dinucleotides in the antisense plants. In contrast, segregants lacking the METI antisense construct and epimutants with a hypermethylated allele of sup (clark kent 3), both of which have active METI genes, showed a higher frequency of methylation of CpG dinucleotides and of asymmetric cytosines. We conclude that METI is the predominant CpG methyltransferase and directly or indirectly affects asymmetric methylation.

Mitochondrial DNA sequences in ancient Australians: Implications for modern human origins.

DNA from ancient human remains provides perspectives on the origin of our species and the relationship between molecular and morphological variation. We report analysis of mtDNA from the remains of 10 ancient Australians. These include the morphologically gracile Lake Mungo 3 [ approximately 60 thousand years (ka) before present] and three other gracile individuals from Holocene deposits at Willandra Lakes (<10 ka), all within the skeletal range of living Australians, and six Pleistocene/early Holocene individuals (15 to <8 ka) from Kow Swamp with robust morphologies outside the skeletal range of contemporary indigenous Australians. Lake Mungo 3 is the oldest (Pleistocene) "anatomically modern" human from whom DNA has been recovered. His mtDNA belonged to a lineage that only survives as a segment inserted into chromosome 11 of the nuclear genome, which is now widespread among human populations. This lineage probably diverged before the most recent common ancestor of contemporary human mitochondrial genomes. This timing of divergence implies that the deepest known mtDNA lineage from an anatomically modern human occurred in Australia; analysis restricted to living humans places the deepest branches in East Africa. The other ancient Australian individuals we examined have mtDNA sequences descended from the most recent common ancestor of living humans. Our results indicate that anatomically modern humans were present in Australia before the complete fixation of the mtDNA lineage now found in all living people. Sequences from additional ancient humans may further challenge current concepts of modern human origins.

The CYP88A cytochrome P450, ent-kaurenoic acid oxidase, catalyzes three steps of the gibberellin biosynthesis pathway.

We have shown that ent-kaurenoic acid oxidase, a member of the CYP88A subfamily of cytochrome P450 enzymes, catalyzes the three steps of the gibberellin biosynthetic pathway from ent-kaurenoic acid to GA(12). A gibberellin-responsive barley mutant, grd5, accumulates ent-kaurenoic acid in developing grains. Three independent grd5 mutants contain mutations in a gene encoding a member of the CYP88A subfamily of cytochrome P450 enzymes, defined by the maize Dwarf3 protein. Mutation of the Dwarf3 gene gives rise to a gibberellin-responsive dwarf phenotype, but the lesion in the gibberellin biosynthesis pathway has not been identified. Arabidopsis thaliana has two CYP88A genes, both of which are expressed. Yeast strains expressing cDNAs encoding each of the two Arabidopsis and the barley CYP88A enzymes catalyze the three steps of the GA biosynthesis pathway from ent-kaurenoic acid to GA(12). Sequence comparison suggests that the maize Dwarf3 locus also encodes ent-kaurenoic acid oxidase.

Control of flowering time by FLC orthologues in Brassica napus.

FLOWERING LOCUS C (FLC) in Arabidopsis encodes a dosage dependent repressor of flowering. We isolated five FLC-related sequences from Brassica napus (BnFLC1-5). Expression of each of the five sequences in Arabidopsis delayed flowering significantly, with the delay in flowering time ranging from 3 weeks to more than 7 months, relative to the flowering time of 3 weeks in untransformed Ler. In the reciprocal experiment, expression of Arabidopsis FLC (AtFLC) in an early flowering B. napus cultivar delayed flowering by 2-6 weeks, confirming the requirement of this gene for floral repression. In B. napus, we show that late flowering and responsiveness to vernalization correlate with the level of BnFLC mRNA expression. The different BnFLC genes show differential expression in leaves, stems and shoot tips, but expression is not detectable in roots. Vernalization dramatically reduces the level of BnFLC transcript and restores early flowering in the winter cultivar Colombus. We conclude that BnFLC genes confer winter requirement in B. napus and account for the major vernalization-responsive flowering time differences in the different cultivars of B. napus in a manner analogous to that of AtFLC in Arabidopsis ecotypes.

The control of flowering by vernalization.

The process by which vernalization, the exposure of a germinating seed or a juvenile plant to a prolonged period of low temperature, promotes flowering in the adult plant has remained a mystery for many years. The recent isolation of one of the key genes involved in vernalization, FLOWERING LOCUS C, has now provided an insight into the molecular mechanism involved, including the role of DNA methylation.

Expression and parent-of-origin effects for FIS2, MEA, and FIE in the endosperm and embryo of developing Arabidopsis seeds.

The promoters of MEA (FIS1), FIS2, and FIE (FIS3), genes that repress seed development in the absence of pollination, were fused to beta-glucuronidase (GUS) to study their activity pattern. The FIS2GUS product is found in the embryo sac, in each of the polar cell nuclei, and in the central cell nucleus. After pollination, the maternally derived FIS2GUS protein occurs in the nuclei of the cenocytic endosperm. Before cellularization of the endosperm, activity is terminated in the micropylar and central nuclei of the endosperm and subsequently in the nuclei of the chalazal cyst. MEAGUS has a pattern of activity similar to that of FIS2GUS, but FIEGUS protein is found in many tissues, including the prepollination embryo sac, and in embryo and endosperm postpollination. The similarity in mutant phenotypes; the activity of FIE, MEA, and FIS2 in the same cells in the embryo sac; and the fact that MEA and FIE proteins interact in a yeast two-hybrid system suggest that these proteins operate in the same system of control of seed development. Maternal and not paternal FIS2GUS, MEAGUS, and FIEGUS show activity in early endosperm, so these genes may be imprinted. When fis2, mea, and fie mutants are pollinated, seed development is arrested at the heart embryo stage. The seed arrest of mea and fis2 is avoided when they are fertilized by a low methylation parent. The wild-type alleles of MEA or FIS2 are not required. The parent-of-origin-determined differential activity of MEA, FIS2, and FIE is not dependent on DNA methylation, but methylation does control some gene(s) that have key roles in seed development.

Molecular strategies for improving waterlogging tolerance in plants.

Plants, like animals, are obligate aerobes, but due to their inability to move, have evolved adaptation mechanisms that enable them to survive short periods of low oxygen supply, such as those occurring after heavy rain or flooding. Crop plants are often grown on soils subject to waterlogging and many are sensitive to waterlogging of the root zone. The combination of unfavourable weather conditions and suboptimal soil and irrigation techniques can result in severe yield losses. The molecular basis of the adaptation to transient low oxygen conditions has not been completely characterized, but progress has been made towards identifying genes and gene products induced during low oxygen conditions. Promoter elements and transcription factors involved in the regulation of anaerobically induced genes have been characterized. In this paper an account is presented of the molecular strategies that have been used in an attempt to increase flooding tolerance of crop plants.

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.

The molecular basis of vernalization: the central role of FLOWERING LOCUS C (FLC).

In Arabidopsis, the MADS-box protein encoded by FLOWERING LOCUS C (FLC) is a repressor of flowering. Vernalization, which promotes flowering in the late-flowering ecotypes and many late-flowering mutants, decreases the level of FLC transcript and protein in the plant. This vernalization-induced reduction in FLC transcript levels is mitotically stable and occurs in all tissues. FLC activity is restored in each generation, as is the requirement of a low-temperature exposure for the promotion of flowering. The level of FLC determines the extent of the vernalization response in the promotion of flowering, and there is a quantitative relationship between the duration of cold treatment and the extent of down-regulation of FLC activity. We conclude that FLC is the central regulator of the induction of flowering by vernalization. Other vernalization-responsive late-flowering mutants, which are disrupted in genes that encode regulators of FLC, are late-flowering as a consequence of their elevated levels of FLC.

Multiple DNA methyltransferase genes in Arabidopsis thaliana.

Methylation of plant DNA occurs at cytosines in any sequence context, and as the Arabidopsis methyltransferase, METI, preferentially methylates cytosines in CG dinucleotides, it is likely that Arabidopsis has other methyltransferases with different target specificities. We have identified five additional genes encoding putative DNA methyltransferases. Three of these genes are very similar to METI throughout the coding region; these genes probably arose by a series of gene duplication events, the most recent giving rise to METIIa and METIIb. METIIa and b are expressed at low levels in vegetative and floral organs and the level of transcripts is not affected by the introduction of a METI antisense transgene, nor do the METII enzymes substitute for the reduced activity of METI in methylating CG dinucleotides. METIII is not essential as it encodes a truncated protein. Two other genes encode a second class of DNA methyltransferase with the conserved motifs characteristic of cytosine methyltransferases, but with little homology to the METI-like methyltransferases through the remainder of the protein. These two methyltransferases are characterized by the presence of a chromodomain inserted within the methyltransferase domain, suggesting that they may be associated with heterochromatin. Both these genes are transcribed at low levels in vegetative and reproductive tissues.

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.