Regulation of sulphur partitioning between primary and secondary metabolism

In the last two decades we have made great progress in improving our air quality by reducing emissions, e.g. of sulphur dioxide. As a consequence, however, less sulphur is falling naturally onto the Earth`s surface and there is a lack of sulphur in the soil. This has become a serious problem for UK agriculture. Sulphur is essential for all living organisms as it is bound in proteins and found in many other compounds vital for life. To make such compounds, plants need to take up sulphate from the soil, reduce it, and incorporate it into amino acids. In addition to these 'primary' compounds, plants synthesize a great variety of other biologically active 'secondary' molecules that are responsible for scent, colour, or the taste of plants. They also help in the defence of the plants against insects, fungi, or bacteria. Some crops, such as oilseed rape, produce a lot of these secondary compounds and therefore require more sulphur for their growth than other plants. We want to know how plants divide the sulphur they take up between the primary and secondary products. We will concentrate on one family of proteins that we believe are responsible for control of sulphur flow. We will create plants where these proteins will no longer be active and plants which will have much more of them. Comparing such plants will enable us to understand how the sulphur is divided between primary and secondary compounds. We want to test, whether plants that save sulphur by producing less of the secondary compounds will be as healthy as normal plants. The results of this project could allow us to develop crop plants that take up and use sulphur more efficiently, reducing the need for fertilisers.

Sulphur is essential for life as a constituent of the amino acids cysteine and methionine and of coenzymes, such as iron-sulphur centres, lipoic acid, or thiamine. In addition, plants synthesise a great variety of secondary metabolites containing sulphur in the oxidised form of sulphate. The best characterised group of such compounds is the glucosinolates, important for plant defence against pathogens, but also of great nutritional value. Sulphation of the desulpho- precursors of the secondary metabolites is catalysed by sulphotransferases that utilise phosphoadenosine phosphosulphate (PAPS) as a sulphate donor. PAPS is synthesised by phosphorylation of adenosine phosphosulphate (APS), a key intermediate in primary sulphate assimilation to cysteine, by APS kinase. Thus, primary and secondary sulphur metabolism is connected by a single enzymatic reaction catalysed by APS kinase. This allows a unique opportunity to study the interactions between primary and secondary sulphur metabolism as nothing is known about the control of partitioning of sulphur between them. The aim of the project is to decipher mechanisms controlling the partitioning of sulphur between primary and secondary metabolism. The project will focus on APS kinase gene family. Differences in kinetic parameters of APS kinase isoforms and their inter- and intracellular localization will be assessed. Furthermore expression analysis of plants subjected to biotic and abiotic stress will address the regulation of mRNA accumulation of the 4 APS kinase isoforms. The expression analysis will be complemented by promoter analysis. Finally, mutant plants with single or multiple knockouts of the individual APK isoforms will be analysed to complete the functional analysis. To study the role of APS kinase in control of flux into secondary sulphur compounds, plants with increased capacity to synthesise PAPS due to overexpression of APS kinase and plants where all 4 APS kinase genes are disrupted or where APS kinase is reduced by an RNAi approach will be analysed. In the different plant lines levels of sulphated secondary compounds, especially glucosinolates, will be compared with accumulation of sulphur-containing primary metabolites. The metabolite analysis will be supplemented by measurements of sulphur fluxes into primary sulphur metabolites (Cys, glutathione) and secondary compounds. Flux analysis of the transgenic plants with modified APS kinase activity and of plants where activity of APS reductase will be modulated by feeding positive and negative regulators will reveal the role of the two enzymes in control of sulphur partitioning between primary and secondary metabolism. By manipulation of APS kinase transgenic plants will be produced with diminished and possibly increased levels of sulphur-containing secondary compounds. Such plants will be analysed for sulphur use efficiency and their resistance against pathogens and environmental stress. This project, therefore, represents the first functional examination of partitioning of sulphur between primary and secondary metabolism. It will increase our understanding of sulphur nutrition in plants for the improvement of sustainable practices in agriculture.