Molecular mechanism and regulation of sulfate assimilation

Molecular mechanisms of regulation of sulfate assimilation

The key step of sulfate assimilation in plants, the two-electron reduction of adenosine 5'-phosphosulfate to sulfite and AMP, is catalyzed by adenosine 5'-phosphosulfate reductase (APR). APR is regulated on the level of mRNA and also post-translationally by thiols and is more susceptible to regulation than other enzymes of the pathway. APR mRNA accumulation and/or enzyme activity are down-regulated by reduced sulfur compounds, nitrogen deficiency, and incubation without CO2 and increased by sulfur deficiency, feeding with reduced forms of nitrogen, carbohydrates, O-acetylserine, buthionine sulfoximine (BSO), phytohormones, or exposure to heavy metals and other environmental stress. In A. thaliana APR possess very high control over the flux through sulfate assimilation. Regulation of APR is, thus, a key to understand the regulation of sulfate assimilation. Other genes of the pathway, however, also contribute to the control. We have generated multiple resources that can now be utilised to address the following important questions:

What are the biological roles of individual isoforms of key enzymes of sulfate assimilation?

We have shown in Arabidopsis that the APR2 isoform of APS reductase has an important role in controlling sulfate accumulation, while the APK1 and APK2 isoforms of APS kinase to be crucial for synthesis of glucosinolates. However, the function of other isoforms of these enzymes as well as other enzymes encoded by multigene families, e.g. ATP sulfurylase or APS reductase, remains to be elucidated. We have collected most of the relevant T-DNA lines and possess the necessary know-how to study the effects of single and multiple gene disruptions on the uptake and assimilation pathways, including flux measurements.

What are the roles of miRNAs in control of plant nutrition?

Several microRNA families have been shown to accumulate in response to P, S, and N starvation. However, with the exception of miR399 in signalling of phosphate deficiency, little is known about their hierarchical position in the regulatory circuits. In collaboration with T. Dalmay at BIO, UEA, we have found that the level of miR395, which targets ATP sulfurylase, is responding to multiple treatments affecting sulfate assimilation, including sulfate starvation. Using plants overexpressing the miRNA or expressing miRNA insensitive forms of the targets we will dissect the function of the miR395 in control of sulfur nutrition.

 

 

 

 

 

 

 

What is the mechanism of demand-driven regulation of plant nutrient assimilation?

We have devised two genetic screens to dissect the demand driven regulation of sulfate assimilation. The first screen used luciferase expressed under control of APR3 promoter as a reporter. The second screen was based on inhibition of root growth by buthionine sulfoximine, a strong inhibitor of glutathione synthesis. We used a combination of map-based cloning transcript based cloning to identify genes underlying the mutant phenotypes. This suite of mutants will be further characterised to identify specific and general components of the APS reductase regulatory network. Among the genes cloned so far was a gene encoding a pentatricopeptide protein that is responsible for splicing of intron 1 of mitochondrial nad7 transcript, linking thus sulfate assimilation with mitochondrial respiration (Figure 2). The identification of transcription factor HY5, involved in control of photomorphogenesis in the screen is especially intriguing since sulfate reduction is light regulated. However, since other metabolic pathways, such as nitrate assimilation, are also regulated by demand, the mutants will be subjected to a broad analysis with the aim of finding specific and general components of signalling pathways and to define a general model of demand-driven regulation.

 

 

 

 

 

 

 

 

Relevant publications:

Relevant publications:
Lee B.-R., Koprivova A., Kopriva S. (2011) Role of HY5 in regulation of sulfate assimilation in Arabidopsis. Plant J., 67, 1042-1054.
Kawashima C.G., Matthewman C.A., Huang S., Lee B.-R., Yoshimoto N., Koprivova A., Rubio-Somoza I., Todesco M., Rathjen T., Saito K., Takahashi H., Dalmay T., Kopriva S. (2011) Interplay of SLIM1 and miR395 in regulation of sulfate assimilation in Arabidopsis. Plant J. 66, 863-876
Koprivova A., Colas des Francs-Small C., Calder G., Mugford S.T., Tanz S., Lee B.-R., Zechmann B., Small I., Kopriva S. (2010) Identification of a pentatricopeptide repeat protein implicated in splicing of intron 1 of mitochondrial nad7 transcripts. J. Biol. Chem. 285, 32192-32199
Davidian J.-C., Kopriva S. (2010) Regulation of sulfate uptake and assimilation - the same or not the same? Mol. Plant 3, 314-325.
Scheerer U., Haensch R., Mendel R.R., Kopriva S., Rennenberg H., Herschbach C. (2010) Sulphur flux through the 1 sulphate assimilation pathway is differently controlled by adenosine 5’-phosphosulphate reductase under stress and in transgenic poplar plants overexpressing γ-ECS, SO or APR. J. Exp. Bot. 61, 609-622
Kopriva S., Mugford S.G., Matthewman C.A., Koprivova A. (2009) Plant sulfate assimilation genes: redundancy vs. specialization. Plant Cell Rep. 28, 1769-1780.
Koprivova A., North K.A., Kopriva S. (2008) Complex signaling network in regulation of sulfate assimilation by salt stress in Arabidopsis roots. Plant Physiol. 146, 1408-1420
Kopriva S., Koprivova A. (2004) Plant adenosine 5'phosphosulfate reductase - the past, the present, and the future. J. Exp. Bot. 55: 1775-1783
Vauclare P., Kopriva S., Fell D., Suter M., Sticher L., von Ballmoos P., Krähenbühl U., Op den Camp R., Brunold C. (2002) Flux control of sulphate assimilation in Arabidopsis thaliana: Adenosine 5’-phosphosulphate reductase is more susceptible to negative control by thiols than ATP sulphurylase. Plant J. 31: 729-740