Regulation of Sulfate Assimilation by Nitrogen in Arabidopsis<sup><a href="#FN1" rid="FN1" class=" fn">1</a></sup>
Abstract
Using Arabidopsis, we analyzed the effect of omission of a nitrogen source and of the addition of different nitrogen-containing compounds on the extractable activity and the enzyme and mRNA accumulation of adenosine 5′-phosphosulfate reductase (APR). During 72 h without a nitrogen source, the APR activity decreased to 70% and 50% of controls in leaves and roots, respectively, while cysteine (Cys) and glutathione contents were not affected. Northern and western analysis revealed that the decrease of APR activity was correlated with decreased mRNA and enzyme levels. The reduced APR activity in roots could be fully restored within 24 h by the addition of 4 mm each of NO3, NH4, or glutamine (Gln), or 1 mmO-acetylserine (OAS). SO4 feeding showed that after addition of NH4, Gln, or OAS to nitrogen-starved plants, incorporation of S into proteins significantly increased in roots; however, glutathione and Cys labeling was higher only with Gln and OAS or with OAS alone, respectively. OAS strongly increased mRNA levels of all three APR isoforms in roots and also those of sulfite reductase, Cys synthase, and serine acetyltransferase. Our data demonstrate that sulfate reduction is regulated by nitrogen nutrition at the transcriptional level and that OAS plays a major role in this regulation.
Several studies have established regulatory interactions between assimilatory sulfate and nitrate reduction in plants (Rennenberg and Bergmann, 1979; Reuveny et al., 1980; Cacco et al., 1983; Brunold and Suter, 1984; Haller et al., 1986; Takahashi and Saito, 1996). The two assimilatory pathways are very similar and well coordinated (Brunold, 1993). Deficiency for one element was shown to repress the other pathway (Reuveny et al., 1980; Neuenschwander et al., 1991). The activities of ATP sulfurylase (ATPS) and adenosine 5′-phosphosulfate (APS) reductase (APR), which has been shown to be identical to APS sulfotransferase (Suter et al., 2000), decreased under nitrogen-deficient conditions in Lemna minor L. and cultured tobacco cells (Reuveny et al., 1980; Brunold and Suter, 1984). At the same time, the addition of nitrate or ammonia to the medium quickly restored the activity of these two enzymes. Later, ATPS was shown to not always be repressed by the absence of nitrogen (Haller et al., 1986), whereas the activity of APR always rapidly decreased under these conditions (Brunold, 1990). Activity of the last enzyme of sulfate assimilation pathway, Cys synthase (CS), can also be critical in varying the flux through the sulfate reduction pathway, since the precursor of Cys, O-acetylserine (OAS), is derived from the carbon and nitrogen assimilation pathways. Accordingly, several studies showed that expression of different isoforms of CS is regulated differently under both sulfur and nitrogen starvation (Takahashi and Saito, 1996; Warrilow and Hawkesford, 1998).
ATPS and APR occupy a central control position of sulfate reduction (Brunold, 1990). Three isoforms of ATPS were cloned from Arabidopsis (Leustek et al., 1994; Klonus et al., 1995; Murillo and Leustek, 1995), all possessing a putative chloroplast-targeting peptide. Also, APR forms a small family of presumably chloroplast localized proteins. Three cDNA clones coding for APR isoforms were obtained from Arabidopsis (Gutierrez-Marcos et al., 1996; Setya et al., 1996). Other enzymes involved in sulfate reduction have already been characterized on a molecular level (Hell et al., 1994; Ruffet et al., 1995; Brühl et al., 1996). The availability of molecular probes now makes it possible to study the regulation of the whole pathway at a molecular level. Takahashi et al. (1997) demonstrated that sulfate-deficiency-induced expression of three genes of the pathway only, the sulfate transporter AST68, APR1, and SAT-1 (Ser acetyltransferase).
We report the effects of nitrogen deficiency and the subsequent addition of different nitrogen sources on APR mRNA and protein accumulation and activity in Arabidopsis shoots and roots. To address the flux through the sulfate assimilation pathway under these conditions, we also determined the incorporation of [S]sulfate into Cys, glutathione (GSH), and proteins.
ACKNOWLEDGMENTS
We thank Dr. T. Leustek and Dr. R. Hell for cDNA probes of APR, CS, and SAT, respectively. We would also like to acknowledge Dr. M. Hawkesford for helpful discussions.
Footnotes
This work was supported by the Swiss National Foundation (grant no. 3149246–96 to C.B.).