Prof Jim Murray
Arabidopsis thaliana is the model plant species we use for our work. It is a rapidly growing plant for which the genome sequence, and many genetic and molecular tools are available.
Molecular Development in Plants
Plant Cell Division and Cell Cycle Control
Cell division in plants is largely concentrated in specialised regions known as meristems, which contain the stem cells. The most important meristems are located at the tips of shoots and roots. We are interested in the mechanisms by which cell division is controlled in plant cells, how it is organised within meristems, and how this changes not only during plant development, but also in response to the environment.
The processes which a cell undergoes in order to divide is termed the cell cycle. Our main interest is in genes that control the entry into the cell cycle - the decision as to whether a cell will divide or not. We cloned a family of genes from plants called D-type cyclins (CYCD), which are related to genes that control the same process in mammals. Further analysis showed, somewhat unexpectedly, that the whole pathway of commitment to cell division is conserved between plants and mammals, although the signals to which cell division responds are, of course, different.
D-type cyclins in both plants and mammals share the property of responding to signals that come from outside the cell. In Arabidopsis, one group of the D-type cyclins called CYCD3 responds to the plant hormone cytokinin, and overexpression of CYCD3 can replace the requirement for exogenous cytokinin (Riou-Khamlichi et al., 1999). Arabidopsis mutants lacking all three CYCD3 genes show reduced cell division and their organs are made of fewer cells (Dewitte et al, 2007).
Plants are unusual in having large numbers of genes controlling the cell cycle, and we have used cell cultures to study their action in the cell cycle, employing biochemical and transcript profiling techniques. This has provided the understanding of the timing of action of genes, and allowed us to progress to analyzing their role in plant development.
We have developed methods to grow cells of Arabidopsis in liquid culture and synchronise their division so that all cells progress through the cell cycle at the same time. The picture shows the nuclei stained with a DNA binding dye, the bright spot being a cell undergoing mitosis.
RNA extracted from synchronized culture cells has been extracted at different times, corresponding to different positions in the cell cycle. This RNA has then been analysed using microarrays to define the genes which have peak expression (red) at different times as cells move through the cell cycle (left to right across picture).
In addition to the normal mitotic cycle, in which the chromosomes are replicated and then shared between two daughter cells, plant cells switch to an alternative cycle called the endocycle, in which DNA is replicated (S phase) but not segregated. Therefore each endocycle round doubles the DNA content of the cell. This switch to endocycles is associated with differentiation of cells and a large increase in volume that drives much of plant growth (compare the meristem and cotyledon cells in the section below). The balance of mitotic cycles and endocycles, and the control of the switch between them, determines how many cells comprise a plant organ and how large those cells are. This control is a major focus of research in the lab.
The lab takes an integrated view of cell division in plants, and studies its control and role at the biochemical and molecular level, in cell suspension cultures and at the developmental level in transgenic plants and mutants.
Systems approaches to Meristem Organisation and Maintenance
Section through the shoot apex of Arabidopsis showing the domed meristem in the centre and two young leaves emerging. The large, expanded cells of the cotyledons (seed leaves) illustrate the difference in size between the meristematic cells (M) and the final expanded cells (C) of mature organs.
Top view of the shoot apical meristem of Arabidopsis in a false colour scanning electron micrograph showing the position of the central stem cell pool in blue, the surrounding organogenic cells in green, and the positions of sequential organ primordia (P1-P8). The next primordium will form at the position indicated by P0.
In order to understand how the behaviour of individual cells is co-ordinated to build complex structures during plant development, we are applying systems analysis approaches. One example is the shoot apical meristem, which forms the tip of shoots. This is a shallow domed structure containing the stem cell niche in the central region, surrounded by an organogenic zone where new organs (leaves or flowers) are initiated. In ongoing projects, we are seeking to understand how cell identity, cell division and cell differentiation are interlinked and coordinated at the molecular level by genetic regulators and the hormones cytokinin and auxin. This work is funded through the EU SY-STEM (Systems Biology of Stem Cell Function in Arabidopsis) network (http://www.sy-stem.ethz.ch/ ) and a European Research Area in Plant Functional Genomics (http://www.erapg.org/everyone ) network on Plant Stem Cells, in which the Cardiff lab is the co-ordinator.
The influence of the environment
Seed germination is a critical point in the plant life cycle, and involves the resumption of metabolism, growth and division. Cells undergoing division can be visualised as they pass through mitosis with a genetic reporter of a mitotic cyclin fused to the GUS gene. Only cells in mitosis stain blue. Here the activation of division in the root meristem during germination is visualised in a synchronized germination experiment investigating how division is resumed as the seed germinates.
A further unique aspect of plant growth is that it responds to the environment. Under conditions of stress- such as low water or high temperature- plants stop growing. With funding from the BBSRC (www.bbsrc.ac.uk ) and Bayer CropScience (http://www.bayercropscience.com/ ), we are seeking to understand how and why they stop growing, and what is happening at the cellular level. Molecular insights into this process and how it is signalled may lead to opportunities to engineer crops with increased stress tolerance and so higher yields.
Molecular biotechnology: Engineering new opportunities
Firefly luciferase and applications of bioluminescence in diagnostics
Firefly luciferase emits light in the presence of its substrate luciferin and ATP, and is widely used as a method of measuring ATP concentrations.
Since all living organisms contain ATP, firefly luciferase it finds widespread application in diagnostic assays for contamination in the pharmaceutical, food processing and related industries as well as for environmental monitoring. The native enzyme is rather labile, and we have successfully engineered the enzyme for improved stability, providing greater utility in a range of assays, and these enzymes have now been commercialized.
Bioluminescent detection of DNA amplification. The lab developed a new patented method called BART- bioluminescent assay in real time to detect DNA amplification. As DNA is amplified (visualised by the upper band appearing on the gel electrophoresis image above), light is emitted and can be readily detected using a camera or other device.
Using the thermostable luciferases, we developed a new bioluminescent method to detect specific nucleic acid sequences known as BART (Bioluminescent Assay in Real-Time). This novel reporter system that permits real-time, quantitative detection of nucleic acids during isothermal DNA amplification without the need for fluorescent reporters and has been commercialized through a spin-out company Lumora, jointly funded by the University of Cambridge and commercial investors. BART works by coupling the generation of pyrophosphate, a by-product of nucleic acid synthesis, to the emission of light from a highly thermostable version of firefly luciferase. The amplification of a specific target nucleic acid sequence therefore becomes linked to a luminescent output from the sample itself (for a detailed explanation of the technology see http://www.lumora.co.uk/Technology.php?view=BART ). Since light is readily measured by simple devices, portable and cheap apparatus can be built to carry out molecular tests that previously were only possible in the laboratory. We are applying this technology to develop methods to track GM foodstuffs with the EU Co-EXTRA consortium (http://www.coextra.eu/ ) as well as detecting pathogenic microorganisms.
Yeast plasmids and applications of yeast
Budding yeast (Saccharomyces cerevisiae) is almost unique in having a small nuclear plasmid that has specific mechanisms that ensure its maintenance. The plasmid appears to be a relatively benign molecular parasite- it is very rarely lost from yeast cells and confers a measurable but slight disadvantage to its host. The plasmid has evolved a transmission mechanism independent of centromere function and a copy number control mechanism. These characteristics have led to the widespread use of the plasmid as a basis for cloning and expression vectors. We are interested in the chromatin modifications that allow the maintenance mechanisms to operate effectively. We also make use of yeast in the development of novel assays for screening for inhibitors of specific gene function with potential roles as drug candidates.
Mr Patrick Hardinge
Dr Jonathon Harrington