The group of Prof. Dr. Mark Stitt uses a systems-oriented approach to look at how biochemical pathways involved in primary carbon and nitrogen metabolism are integrated and regulated, and how they affect plant growth and development. We are developing user-friendly data visualization tools, sensitive high-throughput assays for enzymes and metabolites, and a suite of growth conditions to reveal the impact of changes in the carbon and nutrient status on metabolism, growth and development. Arabidopsis thaliana, tomato and maize are the main plants used in these investigations.
Primary metabolism impinges on all aspects of physiology, making it an excellent starting point to analyse system responses. To reveal how changes in primary metabolism affect growth and environmental homeostasis, plants are grown in what we call "gauntlets" - defined growth conditions under which cryptic phenotypes can be revealed and quantifiable traits scored. Transcripts, enzyme activities, post-translational modifications, and metabolites are then profiled. This multilevel phenotyping approach is being used to systematically analyse the response to changes in the carbon and nutrient supply, to analyse existing mutants, to screen for new mutants and to characterise the changes after altering the expression of selected candidate genes.
Regulation of carbon allocation during the light / dark cycle
The diurnal cycle – the daily alternation between light and dark - provides an excellent starting point to study how metabolism is regulated and coordinated with growth in a fluctuating environment. Growth and metabolism are driven by photosynthesis in the light, whereas in the dark they depend on reserves that have been accumulated in the previous light period. In many plants, starch is the main storage reserve. Starch synthesis and degradation is regulated across a wide range of conditions such that reserves are almost, but not completely, exhausted at the end of the night. This maximises investment in growth, while avoiding starvation during the night. We want to understand how plants achieve this balance.
One approach involves multi-level ’omics’ analyses (Technical Platforms) at many times during diurnal cycles. These have shown that transcription, translation and metabolism respond dynamically to small changes in the carbohydrate status. They have allowed network construction to identify potentially regulatory factors, and the development of models that predict the contribution of sugar-signalling, light-signalling and the circadian clock to the regulation of transcript levels for each individual gene.
This is complemented by using quantitative data about photosynthesis, respiration, metabolite levels, ribosome, transcript and protein levels (see Technical platforms) to model fluxes at different times in the diurnal cycle, to relate the energetic costs of specific cellular growth processes like protein synthesis to the energy available from photosynthesis and respiration, and to model synthesis and turnover rates of proteins
In parallel, we investigate signalling pathways. We are particularly interested in the function of trehalose-6-phosphate, which acts as a sugar-signal to regulate metabolism and development. Here, we use a combination of biochemical and genetic methods to understand how the cellular trehalose-6-phosphate concentration is regulated and what processes it regulates. We are also interested in the role of the biological clock, which plays a key role in the signalling networks that set the rate of starch degradation.
Relationships between the carbohydrate supply and growth
We are taking several approaches to understand how central metabolism is coordinated with growth. We use multilevel ’omics’ analyses (Technical Platforms) to search for metabolic or molecular parameters that correlate with growth across a series of conditions in which different amounts of carbohydrate are available. We term such controlled growth systems ‘Gauntlets’ (Technical Platforms). We also apply these methods in time-resolved analyses in systems where the rate of growth is changing with time. We investigate whether and how the carbohydrate supply regulates developmental transitions.
Protein synthesis is a major and experimentally tractable component of cellular growth. We have developed methods (Technical Platforms) to measure the concentrations of ribosomes and transcripts and their loading into polysomes. We use these quantitative molecular data to model the rates of protein synthesis and estimate the associated energy costs. These can be compared with the measured growth rates, metabolite levels and estimates of metabolic fluxes to obtain quantitative insights into the relationships between metabolism and growth.
Use of Natural Genetic Diversity as a tool to study regulatory networks
Metabolism and growth are regulated by multi-gene networks. Although we frequently use mutants and transgenic plants with altered activities of specific genes, there are limits to what we can learn about networks by perturbations of single genes. Populations of wild accessions or different crop cultivars carry thousands of mutations or polymorphisms that are combined in a different way in each line. This allows us to study the impact of multiple perturbations on metabolic networks. By profiling a large panel of Arabidopsis accessions for many molecular and metabolic traits (Technical Platforms) and cooperating with computational scientists to search for patterns in the resulting data matrices, we are learning which metabolic processes affect biomass production.
This approach can be extended by using detailed information about the genotypes of the plants to identify genetic loci or even individual genes that affect metabolite levels, enzyme activities or biomass production. In addition to using Arabidopsis, we cooperate with geneticists to apply this approach to crops like maize, tomato and melon.
Interaction with N, P, water deficit and temperature
We also investigate how other inputs affect the rate of growth. As with carbohydrates, our first step is to establish growth protocols that allow a moderate and sustained restriction of growth by a particular input (Technical Platforms). We then apply our ’omics’ analyses and investigate the response of the large panel of Arabidopsis accessions to identify general relationships between metabolism and growth in each condition and to identify loci or genes that affect metabolism and biomass production.