Dr. Andrew C. Cuming

Profile

The Centre for Plant Sciences

Research interests

Evolution of plant development; Adaptations in early land plants; Gene targeting

Evolution of gene function:

Our research is centred on the model bryophyte, Physcomitrella patens, a moss that was the first non-flowering plant to have its genome sequenced. It has become a model of choice to investigate the evolution of gene function in plants, due to the powerful tools available for genetic analysis and genetic manipulation. Most remarkably, transgenes can be precisely delivered to predetermined loci within its genome ("gene targeting"), through the integration of transforming DNA by homologous recombination. This occurs with very high efficiency and enables precision engineering of the moss genome. It is a powerful tool for "reverse genetics" that has now been supplemented by the ability to identify randomly generated mutations by the more traditional "forward genetic" approach.

The bryophytes are the most ancient extant lineage of land plants, and retain genetic adaptations that enabled the colonisation of terrestrial habitats by plants approximately 470 milion years ago - an event of planetary significance. The conquest of land by the first plants brought about a major atmospheric oxygenation to levels enabling the subsequent evolution of large land animals. What were the adaptations that enabled previously aquatic plants to succeed in the terrestrial environment? We focus on the ability of plants to survive dehydration: a prerequisite for surviving the transition from water to land. Many bryophytes have the ability to survive complete desiccation in the vegetative state, a trait that is rare among the great diversity of plants that evolved following the initial colonisation. Nevertheless, the majority of modern plants also retain the ability of survive desiccation, although this trait is now restricted to only a small part of their life-cycle, namely in the development of seeds.

Seeds undergo a natural and progressive dehydration culminating in the loss of almost all water from their cells, and they can remain in a dry but viable state for many years (thousands in some cases), only germinating when provided with water. The desiccation tolerance exhibited by seeds is arguably the single most important biological trait for the evolution of our own species, since our ability to harvest and store seed in the dry state from one year to the next supported the development of agriculture - a technology that enabled settlement, population growth and the development of what we now recognise as civilisation.

Desiccation tolerance in seeds is dependent on the accumulation in the cells of protective macromolecules: principally a class of proteins termed "Late Embryogenesis Abundant" (LEA) and sugars that promote vitrification of the cytoplasm. The accumulation of these products is regulated by the plant hormone, Abscisic Acid (ABA), that triggers a programme of gene expression in response to dehydration, promotes seed dormancy and results in the desiccation-tolerant state. From our studies in Physcomitrella, we now know that the ABA-mediated response to dehydration is an ancient one, that is conserved in all land plants. ABA accumulates in tissues subjected to water stress, and binds cytosolic receptor proteins. These undergo a conformational change enabling them to sequester protein phosphatases that normally inhibit the activity of protein kinases. Released from this inhibition, these protein kinases are released to phosphorylate effector proteins, such as the transcription factors that activate gene expression necessary for the acquisition of desiccation tolerance.

Interestingly, the signal transduction pathway leading to desiccation tolerance is even more ancient than the first land plants: the sequencing of the genomes of aquatic algae reveals the presence of homologues of genes encoding the ABA-responsive protein phosphatases, protein kinases and transcription factors in these pre-terrestrial plants, suggesting that the ability to survive dehydration was ancestral to the land plant colonisation. However, algal species do not appear to respond to ABA, indicating that the evolution of an ABA-receptor interaction enabled the recruitment of this pathway to modulate the much wider range of responses to dehydration characteristic of terrestrial species.

We use cross-species genetic complementation of targeted mutants in P. patens to determine whether apparently homologous genes derived from highly divergent species are functionally conserved in the regulation of dehydration tolerance.

Gene targeting

"Gene targeting" defines the delivery of transgenes to predetermined genetic loci. Physcomitrella is unique among plant species in the high efficiency of gene targeting, which is mediated by  recombination of sequences in transforming DNA with homologous sequences in the moss genome. Integration of transgenes into genomes occurs when transforming DNA is captured by endogenous mechanisms for repairing double-strand breaks (DSBs) in genomes, and incorporated into the host genome. Most organisms integrate transgenes randomly, even when extensive sequence homology is present, due to a preference for the repair of DNA-DSBs by  a dominant Non-Homologous End-Joining (NHEJ) repair pathway. In P. patens, the default DNA-DSB repair pathway appears to be the homology-dependent repair (HR) pathway, with the consequence that gene targeting occurs with an efficiency comparable of that in yeast. We use targeted mutagenesis of DNA repair genes to identify the mechanisms that underpin targeted transgene integration in P. patens. Analysis of targeted deletion mutants allows us to identify key genes involved in the process. Analysis of precision-engineered point mutations enables us to mechanistically test the roles of key residues within these genes.

We also offer our extensive experience in Physcomitrella transformation and mutant analysis to offer a targeted mutagenenesis service to researchers who would like to undertake comparative analysis of genes of interest which have homologues in P. patens, but who cannot invest the time and resources necessary to develop the methodology in-house. The service includes the generation of deletion mutants, targeted point mutations, promoter-reporter fusions and targeted "knock-ins, and we welcome enquiries by e-mail to a.c.cuming@leeds.ac.uk. 

Qualifications

• BA 1973, Oxford University;
• PhD 1976, Cambridge University.