Control of sulfur partitioning between primary and secondary metabolism
Sulfur is essential for life as a constituent of the amino acids cysteine and methionine and of coenzymes, such as iron-sulfur centres, lipoic acid, or thiamine. In addition, plants synthesise a great variety of secondary metabolites containing sulfur in the oxidised form of sulfate. The best characterised group of such compounds is the glucosinolates, important for plant defence against pathogens, but also of great nutritional value. Sulfation of the desulfo- precursors of the secondary metabolites is catalysed by sulfotransferases that utilise phosphoadenosine phosphosulfate (PAPS) as a sulfate donor. PAPS is synthesised by phosphorylation of adenosine phosphosulfate (APS), a key intermediate in primary sulfate assimilation to cysteine, by APS kinase. Thus, primary and secondary sulfur metabolism is connected by a single enzymatic reaction catalysed by APS kinase (figure 1).
To assess to what extent APS kinase controls the production of sulphated compounds we analysed the corresponding gene family in Arabidopsis thaliana. Analysis of T-DNA insertion knockout lines for each of the four isoforms did not reveal any phenotypical alterations. However, when all six combinations of double mutants were compared, the apk1 apk2 plants were significantly smaller than wild-type plants. The levels of glucosinolates, a major class of sulphated secondary metabolite, and the sulphated 12-hydroxyjasmonate were reduced approximately 5-fold in apk1 apk2 plants (Fig. 2). Although auxin levels were increased in the apk1 apk2 mutants, as is the case for most plants with compromised glucosinolate synthesis, typical high auxin phenotypes were not observed. The reduction in glucosinolates resulted in increased transcript levels for genes involved in glucosinolate biosynthesis and accumulation of desulphated precursors. It also led to great alterations in sulphur metabolism: the levels of thiols increased in the apk1 apk2 plants (Fig.3). The data indicate that the APK1 and APK2 isoforms of APS kinase play a major role in control of synthesis of secondary sulphated metabolites and are required for normal growth rates.
Since among the genes upregulated in apk1 apk2 mutants were genes encoding components of glucosinolate synthesis as well as primary assimilation (ATP sulfurylase, ATPS), we tested whether promoters of ATPS and APK interact with MYB transcription factors controlling glucosinolate synthesis (Hirai et al., 2007; Gigolashvilli et al., 2007a, 2007b, 2008). A transactivation analysis revealed that promoters of APK1, APK2, ATPS1 and ATPS3 are under control of these factors. Interestingly, also promoters of three APS reductase isoforms, encoding the key step in the reductive primary sulfate assimilation, interact with the MYB factors. The results of the transactivation assays were confirmed by qPCR analysis of plants overexpressing these MYB factors (Yatusevich et al., 2010). Thus, PAPS synthesis is an integral part of GLS biosynthesis network (Figure 4), which also includes genes of primary sulfate assimilation.
We now investigate how the interplay between APS kinase and APS reductase controls the flux through sulfate assimilation and the partitioning of sulfur between primary and secondary metabolism.
Mugford S.G., Lee B.-R., Koprivova A., Matthewman C., Kopriva S. (2011) Control of sulfur partitioning between primary and secondary metabolism. Plant J. 65, 96-105.
Yatusevich R., Mugford S.G., Matthewman C., Gigolashvili T., Frerigmann H., Delaney S., Koprivova A., Flügge U.-I., Kopriva S. (2010) Genes of primary sulfate assimilation are part of the glucosinolate biosynthetic network in Arabidopsis thaliana. Plant J. 62, 1-11.
Mugford S.G., Matthewman C.A., Hill L., Kopriva S. (2010) Adenosine 5’ phosphosulfate kinase is essential for Arabidopsis viability. FEBS Lett. 584, 119-123
Kopriva S., Mugford S.G., Matthewman C.A., Koprivova A. (2009) Plant sulfate assimilation genes: redundancy vs. specialization. Plant Cell Rep. 28, 1769-1780.
Mugford S.G., Yoshimoto N., Reichelt M., Wirtz M., Hill L., Mugford S.T., Nakazato Y., Noji M., Takahashi H., Kramell R., Gigolashvili T., Flügge U.-I., Wasternack C., Gershenzon J., Hell R., Saito K., Kopriva S. (2009) Disruption of Adenosine-5’-Phosphosulphate Kinase in Arabidopsis Reduces Levels of Sulphated Secondary Metabolites. Plant Cell 21, 910-927.