A novel genome-wide approach to understand the genetic basis for morphological diversification of leaves

A key problem in biology is to understand how different organisms come to have different forms. In plants, this variation in form is obvious in the many different leaf shapes one sees during a walk in the park. Leaves are also interesting to study because they play a key role in the food chain being the main photosynthetic organs of land plants and thus responsible for CO2 fixation in terrestrial ecosystems. For these reasons, understanding how diversity in leaf form is generated is of considerable interest to scientists. To study this problem we work with hairy bittercress (Cardamine hirsuta), which is a plant that has leaves fully subdivided into smaller leaflets. The presence of leaflets makes this plant very different to its close relative thale cress (Arabidopsis thaliana), which has entire, undivided leaves. We already know a lot about how an entire leaf shape is produced in thale cress because it is easy to grow and do experiments with. Hairy bittercress is also very easy to work with in the lab, so we use it to understand how leaflets are produced. We now want to understand how the genetic make up of the hairy bittercress differs to that of the thale cress resulting in leaflets being produced in only the former species . Specifically we will test whether hairy bittercress leaves produce leaflets because certain genes in this plant carry different information to thale cress genes. To achieve this we will divide the hairy bittercress genome into many small pieces and introduce each piece of this genome into the thale cress. The few thale cress plants that receive a 'leaflet producing' part of the hairy bittercress genome should now produce leaves that resemble the divided leaves of hairy bittercress. This approach will allow us to isolate 'leaflet producing' genes and find out whether the 'leaflet producing information' resides in the regulatory sequences that control when, where and how much a gene is expressed, or in the protein-coding sequences that determine the biochemical activity of a gene product. Isolation of such 'leaflet producing genes' will form the basis for future research where we will reduce or increase their activity within bitter cress to test to what degree leaflet production is sensitive to alterations of their activity.

We propose to study processes responsible for diversification of leaf form between A.thaliana which bears simple leaves and C.hirsuta which bears dissected leaves. To identify novel loci responsible for diversification of leaf form between these species and investigate the prevalence of cis-regulatory divergence in this process we will transform 35-40kb genomic BAC clones from C.hirsuta into A.thaliana. This will be done using a C.hirsuta genomic library constructed in a transformation-competent vector. Thus, if a gene is expressed differently between C.hirsuta and A.thaliana and this difference in expression is sufficient to generate a difference in leaf form, then a key prediction is that transfer of the whole locus from C.hirsuta into A.thaliana should alter A.thaliana leaf shape. In principle, a similar outcome might also be expected if alterations in coding as opposed to regulatory sequences underpin morphological divergence between the two species, but the very high amino-acid sequence similarity between the two species makes this unlikely. Nevertheless, we will also investigate this possibility since rapid identification of genes where divergence in coding sequences has functional significance is an additional advantage of this approach. Notably, C.hirsuta is amenable to reverse genetics approaches so genes that are identified as sufficient for alteration of leaf form in A.thaliana can then be investigated in C.hirsuta to determine whether they are necessary and sufficient for leaflet formation in their native context, using methodologies we have already described for KNOX genes. The proposed approach is essentially a high throughput screen for loci that contribute to species-specific morphological variation by segmentally transforming the genome of one species into the other. The evolutionary relatedness and high genetic tractability of both A.thaliana and C.hirsuta make these plants excellent systems with which to perform this exciting screen.

otential beneficiaries of this research project and plans to engage effectively with these groups are indicated as follows: i. General public ii. Schoolchildren iii. Policymakers iv. Brassica community including breeders i. General public engagement will center on discussing methodology and societal implications of scientific enquiry in the area of plant development and diversity. We will highlight the necessity to build on strong fundamental research to underpin high quality translational research in plant science. Forums for such events are given in impact plan and include local reading groups organized by the lead PI and occasional public lectures and open event at the botanical gardens. ii. Schoolchildren engagement is achieved through University Open Days and will be enhanced via presentation at St Ebbes priimary school in Oxford iii. Specific policymaker engagement will take place in appropriate forums such as GARNET where the lead PI is a committee member, and the 2020 Joint BBSRC/DFG workshop where the lead PI participated. Policymakers or future policymakers may also be engaged at public events outlined in (i) and ultimately bring this knowledge into better informed discussions and science policy decision making. iv. The information developed here should contribute to building a sound knowledge-base for crucifer development that will inform Brassica crop improvement. Furthermore, we will generate a set of lasting resources (transgenic lines; BAC library ) that in the future should be useful for the comparative study in C.hirsuta and Arabidopsis of growth and development traits that are of agronomic interest. Elucidation of processes that regulate leaflet growth will empower efforts to modify tissue growth for agricultural purposes. These issues will be flagged in presentations the PI will offer to present, for example at the annual Brassica Research Community and Oilseed Rape Genetic Improvement Network meetings, on the lab website and via ISIS innovation: the University technology transfer company.