Stephen Pearce

Molecular Characterization of Rht-1 Dwarfing Genes in Hexaploid Wheat

The introduction of the Reduced height (Rht)-B1b and Rht-D1b semidwarfing genes led to impressive increases in wheat (Triticum aestivum) yields during the Green Revolution. The reduction in stem elongation in varieties containing these alleles is caused by a limited response to the phytohormone gibberellin (GA), resulting in improved resistance to stem lodging and yield benefits through an increase in grain number. Rht-B1 and Rht-D1 encode DELLA proteins, which act to repress GA-responsive growth, and their mutant alleles Rht-B1b and Rht-D1b are thought to confer dwarfism by producing more active forms of these growth repressors. While no semidwarfing alleles of Rht-A1 have been identified, we show that this gene is expressed at comparable levels to the other homeologs and represents a potential target for producing novel dwarfing alleles. In this study, we have characterized additional dwarfing mutations in Rht-B1 and Rht-D1. We show that the severe dwarfism conferred by Rht-B1c is caused by an intragenic insertion, which results in an in-frame 90-bp insertion in the transcript and a predicted 30-amino acid insertion within the highly conserved amino-terminal DELLA domain. In contrast, the extreme dwarfism of Rht-D1c is due to overexpression of the semidwarfing Rht-D1b allele, caused by an increase in gene copy number. We show also that the semidwarfing alleles Rht-B1d and Rht-B1e introduce premature stop codons within the amino-terminal coding region. Yeast two-hybrid assays indicate that these newly characterized mutations in Rht-B1 and Rht-D1 confer “GA-insensitive” dwarfism by producing DELLA proteins that do not bind the GA receptor GA INSENSITIVE DWARF1, potentially compromising their targeted degradation.

Regulation of Freezing Tolerance and Flowering in Temperate Cereals: The VRN-1 Connection

In winter wheat (Triticum spp.) and barley (Hordeum vulgare) varieties, long exposures to nonfreezing cold temperatures accelerate flowering time (vernalization) and improve freezing tolerance (cold acclimation). However, when plants initiate their reproductive development, freezing tolerance decreases, suggesting a connection between the two processes. To better understand this connection, we used two diploid wheat (Triticum monococcum) mutants, maintained vegetative phase (mvp), that carry deletions encompassing VRN-1, the major vernalization gene in temperate cereals. Homozygous mvp/mvp plants never flower, whereas plants carrying at least one functional VRN-1 copy (Mvp/−) exhibit normal flowering and high transcript levels of VRN-1 under long days. The Mvp/− plants showed reduced freezing tolerance and reduced transcript levels of several cold-induced C-REPEAT BINDING FACTOR transcription factors and COLD REGULATED genes (COR) relative to the mvp/mvp plants. Diploid wheat accessions with mutations in the VRN-1 promoter, resulting in high transcript levels under both long and short days, showed a significant down-regulation of COR14b under long days but not under short days. Taken together, these studies suggest that VRN-1 is required for the initiation of the regulatory cascade that down-regulates the cold acclimation pathway but that additional genes regulated by long days are required for the down-regulation of the COR genes. In addition, our results show that allelic variation in VRN-1 is sufficient to determine differences in freezing tolerance, suggesting that quantitative trait loci for freezing tolerance previously mapped on this chromosome region are likely a pleiotropic effect of VRN-1 rather than the effect of a separate closely linked locus (FROST RESISTANCE-1), as proposed in early freezing tolerance studies.

Separating homeologs by phasing in the tetraploid wheat transcriptome

BackgroundThe high level of identity among duplicated homoeologous genomes in tetraploid pasta wheat presents substantial challenges for de novo transcriptome assembly. To solve this problem, we develop a specialized bioinformatics workflow that optimizes transcriptome assembly and separation of merged homoeologs. To evaluate our strategy, we sequence and assemble the transcriptome of one of the diploid ancestors of pasta wheat, and compare both assemblies with a benchmark set of 13,472 full-length, non-redundant bread wheat cDNAs.ResultsA total of 489 million 100 bp paired-end reads from tetraploid wheat assemble in 140,118 contigs, including 96% of the benchmark cDNAs. We used a comparative genomics approach to annotate 66,633 open reading frames. The multiple k-mer assembly strategy increases the proportion of cDNAs assembled full-length in a single contig by 22% relative to the best single k-mer size. Homoeologs are separated using a post-assembly pipeline that includes polymorphism identification, phasing of SNPs, read sorting, and re-assembly of phased reads. Using a reference set of genes, we determine that 98.7% of SNPs analyzed are correctly separated by phasing.ConclusionsOur study shows that de novo transcriptome assembly of tetraploid wheat benefit from multiple k-mer assembly strategies more than diploid wheat. Our results also demonstrate that phasing approaches originally designed for heterozygous diploid organisms can be used to separate the close homoeologous genomes of tetraploid wheat. The predicted tetraploid wheat proteome and gene models provide a valuable tool for the wheat research community and for those interested in comparative genomic studies.

Effect of the down-regulation of the high Grain Protein Content (GPC) genes on the wheat transcriptome during monocarpic senescence

BackgroundIncreasing the nutrient concentration of wheat grains is important to ameliorate nutritional deficiencies in many parts of the world. Proteins and nutrients in the wheat grain are largely derived from the remobilization of degraded leaf molecules during monocarpic senescence. The down-regulation of the NAC transcription factor Grain Protein Content (GPC) in transgenic wheat plants delays senescence (>3 weeks) and reduces the concentration of protein, Zn and Fe in the grain (>30%), linking senescence and nutrient remobilization.Based on the early and rapid up-regulation of GPC in wheat flag leaves after anthesis, we hypothesized that this transcription factor is an early regulator of monocarpic senescence. To test this hypothesis, we used high-throughput mRNA-seq technologies to characterize the effect of the GPC down-regulation on the wheat flag-leaf transcriptome 12 days after anthesis. At this early stage of senescence GPC transcript levels are significantly lower in transgenic GPC-RNAi plants than in the wild type, but there are still no visible phenotypic differences between genotypes.ResultsWe generated 1.4 million 454 reads from early senescing flag leaves (average ~350 nt) and assembled 1.2 million into 30,497 contigs that were used as a reference to map 145 million Illumina reads from three wild type and four GPC-RNAi plants. Following normalization and statistical testing, we identified a set of 691 genes differentially regulated by GPC (431 ≥ 2-fold change). Transcript level ratios between transgenic and wild type plants showed a high correlation (R = 0.83) between qRT-PCR and Illumina results, providing independent validation of the mRNA-seq approach. A set of differentially expressed genes were analyzed across an early senescence time-course.ConclusionsMonocarpic senescence is an active process characterized by large-scale changes in gene expression which begins considerably before the appearance of visual symptoms of senescence. The mRNA-seq approach used here was able to detect small differences in transcript levels during the early stages of senescence. This resulted in an extensive list of GPC-regulated genes, which includes transporters, hormone regulated genes, and transcription factors. These GPC-regulated genes, particularly those up-regulated during senescence, provide valuable entry points to dissect the early stages of monocarpic senescence and nutrient remobilization in wheat.

Divergent functions of orthologous NAC transcription factors in wheat and rice

The wheat GPC-B1 gene located on chromosome 6B is an early regulator of senescence and affects remobilization of protein and minerals to the grain. GPC-B1 is a NAC transcription factor and has a paralogous copy on chromosome 2B in wheat, GPC-B2. The closest rice homolog to both wheat GPC genes is Os07g37920 which is located on rice chromosome 7 and is colinear with GPC-B2. Since rice is a diploid species with a sequenced genome, we initiated the study of Os07g37920 to develop a simpler model to study senescence and mineral remobilization in cereals. We developed eleven independent RNA interference transgenic rice lines (Os07g37920-RNAi) and 10 over-expressing transgenic lines (Os07g37920-OE), but none of them showed differences in senescence. Transgenic Os07g37920-RNAi rice plants had reduced proportions of viable pollen grains and were male-sterile, but were able to produce seeds by cross pollination. Analysis of the flower morphology of the transgenic rice plants showed that anthers failed to dehisce. Transgenic Os07g37920-OE lines showed no sterility or anther dehiscence problems. Os07g37920 transcript levels were higher in stamens compared to leaves and significantly reduced in the transgenic Os07g37920-RNAi plants. Wheat GPC genes showed the opposite transcription profile (higher transcript levels in leaves than in flowers) and plants carrying knock-out mutations of all GPC-1 and GPC-2 genes exhibited delayed senescence but normal anther dehiscence and fertility. These results indicate a functional divergence of the homologous wheat and rice NAC genes and suggest the need for separate studies of the function and targets of these transcription factors in wheat and rice.

Characterization of Flowering Locus T1 (FT1) gene in Brachypodium and wheat

The phase transition from vegetative to reproductive growth is a critical event in the life cycle of flowering plants. FLOWERING LOCUS T (FT) plays a central role in the regulation of this transition by integrating signals from multiple flowering pathways in the leaves and transmitting them to the shoot apical meristem. In this study, we characterized FT homologs in the temperate grasses Brachypodium distachyon and polyploid wheat using transgenic and mutant approaches. Downregulation of FT1 by RNAi was associated with a significant downregulation of the FT-like genes FT2 and FT4 in Brachypodium and FT2 and FT5 in wheat. In a transgenic wheat line carrying a highly-expressed FT1 allele, FT2 and FT3 were upregulated under both long and short days. Overexpression of FT1 caused extremely early flowering during shoot regeneration in both Brachypodium and hexaploid wheat, and resulted in insufficient vegetative tissue to support the production of viable seeds. Downregulation of FT1 transcripts by RNA interference (RNAi) resulted in non-flowering Brachypodium plants and late flowering plants (2–4 weeks delay) in wheat. A similar delay in heading time was observed in tetraploid wheat plants carrying mutations for both FT-A1 and FT-B1. Plants homozygous only for mutations in FT-B1 flowered later than plants homozygous only for mutations in FT-A1, which corresponded with higher transcript levels of FT-B1 relative to FT-A1 in the early stages of development. Taken together, our data indicate that FT1 plays a critical role in the regulation of flowering in Brachypodium and wheat, and that this role is associated with the simultaneous regulation of other FT-like genes. The differential effects of mutations in FT-A1 and FT-B1 on wheat heading time suggest that different allelic combinations of FT1 homoeologs could be used to adjust wheat heading time to improve adaptation to changing environments.

Exogenous gibberellins induce wheat spike development under short days only in the presence of VERNALIZATION1.

Long-day up-regulation of GA biosynthesis in the wheat apices is required in combination with the meristem identity gene VRN1 for normal spike development. The activation of the meristem identity gene VERNALIZATION1 (VRN1) is a critical regulatory point in wheat (Triticum spp.) flowering. In photoperiod-sensitive wheat varieties, VRN1 is expressed only under long days (LDs), but mutants carrying deletions in a regulatory element in its promoter show VRN1 transcription and early spike development under short days (SDs). However, complete spike development is delayed until plants are transferred to LDs, indicating the existence of an additional regulatory mechanism dependent on LDs. We show here that exogenous gibberellin (GA) application accelerates spike development under SDs, but only in wheat lines expressing VRN1. The simultaneous presence of GA and VRN1 results in the up-regulation of the floral meristem identity genes SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1-1 and LEAFY, whereas inhibition of GA biosynthesis with paclobutrazol precludes the LD induction of these two genes. The inductive role of GA on wheat flowering is further supported by the up-regulation of GA biosynthetic genes in the apices of plants transferred from SDs to LDs and in photoperiod-insensitive and transgenic wheat plants with increased FLOWERING LOCUS T transcription under SDs. The up-regulation of GA biosynthetic genes was not observed in the leaves of the same genetic stocks. Based on these observations, we propose a model in which FLOWERING LOCUS T is up-regulated in the leaves under LDs and is then transported to the shoot apical meristem, where it simultaneously induces the expression of VRN1 and GA biosynthetic genes, which are both required for the up-regulation of the early floral meristem genes SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1-1 and LEAFY and the timely development of the wheat spike.

Heterologous expression and transcript analysis of gibberellin biosynthetic genes of grasses reveals novel functionality in the GA3ox family

BackgroundThe gibberellin (GA) pathway plays a central role in the regulation of plant development, with the 2-oxoglutarate-dependent dioxygenases (2-ODDs: GA20ox, GA3ox, GA2ox) that catalyse the later steps in the biosynthetic pathway of particularly importance in regulating bioactive GA levels. Although GA has important impacts on crop yield and quality, our understanding of the regulation of GA biosynthesis during wheat and barley development remains limited. In this study we identified or assembled genes encoding the GA 2-ODDs of wheat, barley and Brachypodium distachyon and characterised the wheat genes by heterologous expression and transcript analysis.ResultsThe wheat, barley and Brachypodium genomes each contain orthologous copies of the GA20ox, GA3ox and GA2ox genes identified in rice, with the exception of OsGA3ox1 and OsGA2ox5 which are absent in these species. Some additional paralogs of 2-ODD genes were identified: notably, a novel gene in the wheat B genome related to GA3ox2 was shown to encode a GA 1-oxidase, named as TaGA1ox-B1. This enzyme is likely to be responsible for the abundant 1β-hydroxylated GAs present in developing wheat grains. We also identified a related gene in barley, located in a syntenic position to TaGA1ox-B1, that encodes a GA 3,18-dihydroxylase which similarly accounts for the accumulation of unusual GAs in barley grains. Transcript analysis showed that some paralogs of the different classes of 2-ODD were expressed mainly in a single tissue or at specific developmental stages. In particular, TaGA20ox3, TaGA1ox1, TaGA3ox3 and TaGA2ox7 were predominantly expressed in developing grain. More detailed analysis of grain-specific gene expression showed that while the transcripts of biosynthetic genes were most abundant in the endosperm, genes encoding inactivation and signalling components were more highly expressed in the seed coat and pericarp.ConclusionsThe comprehensive expression and functional characterisation of the multigene families encoding the 2-ODD enzymes of the GA pathway in wheat and barley will provide the basis for a better understanding of GA-regulated development in these species. This analysis revealed the existence of a novel, endosperm-specific GA 1-oxidase in wheat and a related GA 3,18-dihydroxylase enzyme in barley that may play important roles during grain expansion and development.

Large deletions in the CBF gene cluster at the Fr-B2 locus are associated with reduced frost tolerance in wheat

Abstract Wheat plants which are exposed to periods of low temperatures (cold acclimation) exhibit increased survival rates when they are subsequently exposed to freezing temperatures. This process is associated with large-scale changes in the transcriptome which are modulated by a set of tandemly duplicated C-repeat Binding Factor (CBF) transcription factors located at the Frost Resistance-2 (Fr-2) locus. While Arabidopsis has three tandemly duplicated CBF genes, the CBF family in wheat has undergone an expansion and at least 15 CBF genes have been identified, 11 of which are present at the Fr-2 loci on homeologous group 5 chromosomes. We report here the discovery of three large deletions which eliminate 6, 9, and all 11 CBF genes from the Fr-B2 locus in tetraploid and hexaploid wheat. In wild emmer wheat, the Fr-B2 deletions were found only among the accessions from the southern sub-populations. Among cultivated wheats, the Fr-B2 deletions were more common among varieties with a spring growth habit than among those with a winter growth habit. Replicated freezing tolerance experiments showed that both the deletion of nine CBF genes in tetraploid wheat and the complete Fr-B2 deletion in hexaploid wheat were associated with significant reductions in survival after exposure to freezing temperatures. Our results suggest that selection for the wild-type Fr-B2 allele may be beneficial for breeders selecting for varieties with improved frost tolerance.

WheatExp: an RNA-seq expression database for polyploid wheat

BackgroundFor functional genomics studies, it is important to understand the dynamic expression profiles of transcribed genes in different tissues, stages of development and in response to environmental stimuli. The proliferation in the use of next-generation sequencing technologies by the plant research community has led to the accumulation of large volumes of expression data. However, analysis of these datasets is complicated by the frequent occurrence of polyploidy among economically-important crop species. In addition, processing and analyzing such large volumes of sequence data is a technical and time-consuming task, limiting their application in functional genomics studies, particularly for smaller laboratories which lack access to high-powered computing infrastructure. Wheat is a good example of a young polyploid species with three similar genomes (97 % identical among homoeologous genes), rapidly accumulating RNA-seq datasets and a large research community.DescriptionWe present WheatExp, an expression database and visualization tool to analyze and compare homoeologue-specific transcript profiles across a broad range of tissues from different developmental stages in polyploid wheat. Beginning with publicly-available RNA-seq datasets, we developed a pipeline to distinguish between homoeologous transcripts from annotated genes in tetraploid and hexaploid wheat. Data from multiple studies is processed and compiled into a database which can be queried either by BLAST or by searching for a known gene of interest by name or functional domain. Expression data of multiple genes can be displayed side-by-side across all expression datasets providing immediate access to a comprehensive panel of expression data for specific subsets of wheat genes.ConclusionsThe development of a publicly accessible expression database hosted on the GrainGenes website - http://wheat.pw.usda.gov/WheatExp/ - coupled with a simple and readily-comparable visualization tool will empower the wheat research community to use RNA-seq data and to perform functional analyses of target genes. The presented expression data is homoeologue-specific allowing for the analysis of relative contributions from each genome to the overall expression of a gene, a critical consideration for breeding applications. Our approach can be expanded to other polyploid species by adjusting sequence mapping parameters according to the specific divergence of their genomes.