Sulfate resupply accentuates protein synthesis in coordination with nitrogen metabolism in sulfur deprived Brassica napus

Highlights

• N and S assimilation was assessed by SO₄²⁻ resupply followed by S-deprivation.

• S-resupply activated rapidly the activity of NR, GS and OASTL.

• ATPS and APR activity does not match with the proteins increased by S-resupply.

• S-resupply increased the incorporation of ¹⁵N and ³⁴S to amino acids and proteins.

• S-resupply accelerates nitrate assimilation mainly for protein synthesis.

Abstract

To investigate the regulatory interactions between S assimilation and N metabolism in Brassica napus, de novo synthesis of amino acids and proteins was quantified by ¹⁵N and ³⁴S tracing, and the responses of transporter genes, assimilatory enzymes and metabolites pool involving in nitrate and sulfate metabolism were assessed under continuous sulfur supply, sulfur deprivation and sulfate resupply after 3 days of sulfur (S) deprivation. S-deprived plants were characterized by a strong induction of sulfate transporter genes, ATP sulfurylase (ATPS) and adenosine 5′-phosphosulfate reductase (APR), and by a repressed activity of nitrate reductase (NR) and glutamine synthetase (GS). Sulfate resupply to the S-deprived plants strongly increased cysteine, amino acids and proteins concentration. The increase in sulfate and cysteine concentration caused by sulfate resupply was not matched with the expression of sulfate transporters and the activity of ATPS and APR which were rapidly decreased by sulfate resupply. A strong induction of O-acetylserine(thiol)lyase (OASTL), NR and GS upon sulfate resupply was accompanied with the increase in cysteine, amino acids and proteins pool. Sulfate resupply resulted in a strong increase in de novo synthesis of amino acids and proteins, as evidenced by the increases in N and S incorporation into amino acids (1.8- and 2.4-fold increase) and proteins (2.2-and 6.3-fold increase) when compared to S-deprived plants. The results thus indicate that sulfate resupply followed by S-deprivation accelerates nitrate assimilation for protein synthesis.

Introduction

Oilseed rape (Brassica napus L.) has a higher critical N and S demand (Berry et al., 2010) and requires relatively high amount of other nutrients (Rathke et al., 2006) when compared to cereals. In recent decades, the reduction of industrial S emissions to the atmosphere and the subsequent reduction of deposition on the soil have increased the incidence of S-limitation in oilseed rape in many agricultural areas (McGrath and Zhao, 1995). The limitation of sulfur supply generally induces multiple responses facilitating sulfate uptake efficiency at the whole plant level. For instance, in sulfate deprived plants, there is strongly enhanced expression of various members of the sulfate transporters (Hawkesford, 2003, Buchner et al., 2004, Kataoka et al., 2004, Koralewska et al., 2009). The uptake, distribution and reduction of sulfate in plants are driven presumably by the sulfur demand for growth (Anderson and Fitzgerald, 2003) and highly coordinated with exogenous sulfur supply (Hawkesford, 2003, Buchner et al., 2004). However, there was apparently no strict and direct shoot to root signaling for the regulation of sulfate uptake and transport to the shoot in relation to the variation in sulfur supply (Hawkesford and De Kok, 2006). The relationship between sulfate content and expression of the sulfate transporters is ambiguous (Koralewska et al., 2009). For example, the increase in the overall capacity of sulfate uptake by root is limited (Hawkesford, 2003, Buchner et al., 2004, Lee et al., 2013, Lee et al., 2014), although the expression of sulfate transporters is strongly enhanced under S-limitation (Buchner et al., 2004, Kataoka et al., 2004, Honsel et al., 2012).

Numerous studies have suggested regulatory interactions between S assimilation and N metabolism in higher plants (Takahashi and Saito, 1996, Koprivova et al., 2000, Hesse et al., 2004, Carfagna et al., 2011). Based on the results with cultured tobacco cells, it has long been proposed a scheme that integrates the regulation of sulfate and nitrate assimilation, in which each pathway is down-regulated by its own end products and up-regulated by the products of the other pathway (Reuveny and Filner, 1977). These interactions ensure a coordinated flow of the two essential elements because plants use most assimilated sulfate and nitrate for protein synthesis at a relatively stable N to S molar ratio (Rennenberg, 1984). Nitrate induces genes involved in sulfate uptake and assimilation in higher plants, increasing the sulfate assimilation rates (Ehira et al., 2003, Hesse et al., 2004). The activities of ATP sulfurylase (ATPS), Adenosine 5′-phosphosulfate reductase (APR), and O-acetylserine(thiol)lyase (OASTL) decreased under N-deficiency in Lemna minor (Brunold and Suter, 1984) and cultured tobacco cells (Reuveny and Filner, 1977), and restored when nitrate or ammonia were resupplied (Reuveny et al., 1980). A decrease in APR activity in N-deficient plants correlated with a decrease of mRNA abundance level and enzyme activity involved in sulfate assimilation in Arabidopsis (Koprivova et al., 2000). Under S-deficiency conditions, an increase in soluble nitrogen content including nitrate and amides, and a reduction in the internal S pool have been observed (Lee et al., 2013). In addition, the mRNA of the mitochondrial isoform of OASTL was increased in N-deprived spinach plants (Takahashi and Saito, 1996). The significance of thiol compounds as regulating signals in the control of APR activity and expression has been documented (Kopriva and Koprivova, 2003, Durenkamp et al., 2007, Koralewska et al., 2009). However, it is still contentious whether sulfate itself, pool of sulfate or sulfate/nitrate metabolites (e.g. cysteine, glutathione, amino acids and proteins) acts as regulatory signal in the expression of genes and enzymes involved in sulfate and nitrate uptake and their further metabolism or vice versa.

Taken together, we hypothesized that sulfate resupply to S-deprived plants has a strong influence on amino acids and protein synthesis derived from newly absorbed nitrate and sulfate (de novo synthesis) with regulatory interactions between S assimilation and N metabolism. To test this hypothesis, direct quantification of de novo synthesis of amino acids and proteins was done by tracing ¹⁵N and ³⁴S. In addition, the expression of nitrate of sulfate and nitrate assimilatory enzymes, and the concentration of S- and N-reduced compounds were assessed with three different treatments: continuous sulfur supply (control, +S/+S), continuous sulfur deprivation (−S/−S) and sulfate resupply after 3 days of sulfur deprivation (−S/+S).