Overview

A major focus of our work is in the control of growth. We are interested in how the characteristic final size of plant cells, organs and seeds is set and influenced by the supply of nutrients. We use genetics, biochemistry and physiology in the experimental plant Arabidopsis thaliana in this research. Our work has identified several new genes and processes that contribute to the regulation of cell expansion, the final size of organs and seeds, and the integration of growth with available nutrient supplies. A long- term aim is to use this knowledge to enhance yield in crop plants.

Another focus of our work is on structural genomics of plants. This work involves the sequencing, assembly and analysis of plant genes, defining their relationships and their physical locations on chromosomes, and revealing how evolution and domestication shape plant genomes. This work provides key foundations for genetic analysis, genome– led breeding and systems- level analysis such as gene expression and protein interactions. We have worked on the Arabidopsis and rice genomes in the past, and most recently completed analysis of the compact genome of Brachypodium distachyon, a member of the Pooideae subfamily of grasses. This permitted three-way comparison with the genomes of sorghum (Pancoideae) and rice (Erhardtoideae) that revealed at the whole genome level conserved syntenic blocks of genes across diverse grass species. This provides a foundation for analysis of the larger more complex genomes of other pooid grasses, particularly the Triticeae.  Our current work aims to create useful gene sequences from hexaploid wheat using whole genome shotgun sequencing, to lay the foundations for completing the genome sequence, and contributing to long-range physical mapping of chromosomes and the complete genome.

 

Wheat Genomic Sequencing

Dr Fu-Hao Lu, Mr Neil McKenzie

BBSRC-funded Strategic LOLA project “Triticeae Genomics for Sustainable Agriculture”, jointly with The Genome Analysis Centre, the European Bioinformatics Institute and Rothamsted Research. Matt Clark and Mike Bevan Co-Principal Investigators.

Generating complete and accurate assemblies of wheat genomes is a centrally important scientific and strategic goal, but their large sizes and hexaploid complexity have been major technical barriers. In this joint project new long-read Illumina sequencing technologies have been coupled with PCR-free sequencing templates and novel mate-pair and jumping libraries to generate sequencing resources. These have been assembled into near complete wheat genome sequences and scaffolded to create long arrays of wheat genes. The major challenges are to continue to extend scaffold lengths and to generate efficiently the sequences of several wheat genomes of scientific and agricultural interest.

In this project our group is developing transcriptome resources from different wheat lines and progenitor species for gene annotation and studies of gene expression. In order to improve and assess genome scaffolding we created a Fosill library (Williams et al Genome Research 22:2241) in the vector pFosill4. This library of 55m provides 250bp PE reads separated by an average of 37.5kb, covering the genome to a depth of about 1.5x. Although this coverage is low, it is sufficient to triple scaffold N50 sizes. The precise jump distance also provides a way to assess independently errors in assembly and scaffolding.

The Figure shows the distribution of the wheat Fosill libraries on sequenced wheat chromosome 3B. Peaks are repeats that are separated by 40kb.

 

INvestigating TRiticeae EPIgenomes for Domestication (INTREPID).

Dr Fu-Hao Lu, Mr Neil McKenzie

This collaborative project is funded by the ERA-CAPS programme.

The partners are Dr Anthony Hall (now at TGAC, Norwich), Dr Klaus Mayer (MIPS, Munich), and Drs Richard McCombie and Robert Martienssen (CSHL, New York).

This project is generating knowledge about DNA methylation patterns in the wheat genome, about how these may vary in different wheat lines, and how DNA methylation patterns change during the formation of new hybrids. The key role of DNA methylation in silencing the activities of transposons must be especially important in the wheat genome, which contains 7,249,022 Class I retroelements comprising 67% of the wheat genome. The extent to which cross-talk between the three genomes in bread wheat involves epigenetic modification to balance gene expression is an intriguing question. Also, the extent and consequences of epigenetic reprogramming in the germline (Colarco et al Cell 151:194), particularly during the formation of new wheat hybrids, is unknown in wheat. Currently the epigenome of Chinese Spring, the genomic reference line, is being determined using bisulphite sequencing of leaf tissue, anthers, embryos and endosperm tissue. Small RNAs are also being sampled. Exome capture is being used to sample methylation of the gene space of multiple wheat lines, including breeding varieties.

 

Organ size control in Arabidopsis and Brassicas

Dr Hui Dong, Dr Charlotte Miller, Dr Benguo Gu, Ms Rachel Prior (BBSRC DTP Student), Ms Caroline Smith, Mr Neil McKenzie

This work is funded by the BBSRC, the Newton Fund, the ERA-CAPs project ABCEED, and by the JIC Doctoral Training Partnership.

The mechanisms establishing the final sizes of organs such as leaves and seeds are not very well understood, despite the fundamental biological importance of size and allometry. In plants studying the growth of determinate organs such as leaves and petals has some advantages as growth is in essential two dimensions and does not involve cell movement or apoptosis. We have identified a new mechanism of growth control in Arabidopsis that influences the duration of cell proliferation during organogenesis. An ubiquitin-activated peptidase functions to limit the duration of cell proliferation during leaf and petal growth by the concerted cleavage of proteins that promote proliferation and inhibit endoreduplication. We hypothesise that this mechanism may function as a checkpoint to control the transition from mitotic proliferation to endoreduplication, cell expansion and differentiation. Current work focuses on identifying more substrates of the peptidase, and the cellular location and consequences of peptidase cleavage of substrates. For this work we have teamed up with Veronica Grieneisen and Stan Maree at the JIC to use segmentation analyses to define all cells, their location and shape in a growing leaf. This provides a foundation for understanding peptidase function at a cellular level and for generating quantitative models of growth control by the peptidase. In this work we have developed some useful material for studying ubiquitin in plants. Please contact michael.bevan@jic.ac.uk for further information.

The image on the left is of an 8 day old Arabidopsis leaf expressing an epidermal- specific cell membrane marker fused to GFP. This marks the outlines of cells in the outer layer of the leaf. The image on the right is an example of segmentation, in which the intensity image from confocal microscopy is analysed computationally to recognise cell boundaries. In this way the total number of cells, their location and shape can be analysed during leaf growth.

Association genetic analyses in a panel of Brassica napus (oilseed rape) accessions identified an association between a locus and its gene expression (GEM) patterns and seed yield. Molecular characterisation of the gene and its expression in B. napus accessions and Arabidopsis knock-out and over-expression lines has identified a new role for a ubiquitin E3 ligase in coordinating gene expression in the testa and embryo, leading to increased protein and lipid levels and reduced testa pigmentation.

The Figure shows associations between SNPs (upper panel) and gene expression levels (lower panel) and seed weight and yield in a panel of 104 B. napus lines. These associations identified genetic variation controlling the expression of a gene encoding and E3 ubiquitin ligase.

 

Control of nitrate uptake and assimilation

Dr Jingkun Ma

This work is funded by a BBSRC project.

Plant growth is dependent on external sources of minerals such as nitrate and phosphate. These are found at highly varying levels in the environment, and plants have adapted to this variation through the evolution of uptake and assimilation mechanisms that can adjust to external nutrient levels. In agriculture high levels of external nitrate is used to promote growth, but this is expensive and environmentally damaging. By understanding how nitrate is sensed in plants, and how its assimilation is linked to the availability of necessary metabolites produced by photosynthesis, we hypothesise that it might be possible to engineer the regulation of nitrate uptake to optimise the use of nitrate fertilizers. We have identified the transcription factor NPL7 (Marchive et al Nature Comms 4:1713) as a nexus in nitrate and photosynthate regulation. Current research uses mass spectrometry to identify post-translational modifications to NLP7 in response to nitrate and sugar and to identify interacting proteins. Genetic analyses of these modifications and interacting proteins are providing new insights into nitrate-mediated gene expression.