Research articleSulfate resupply accentuates protein synthesis in coordination with nitrogen metabolism in sulfur deprived Brassica napus
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).
Section snippets
Plant culture, treatment and isotope labeling
Surface-sterilized seeds of B. napus L. cv Capitol were germinated in soil bed. Seedlings at the trifoliate stage were transplanted to 3 L plastic pot and grown with hydroponic nutrient solution (Lee et al., 2013). The nutrient solution was continuously aerated and renewed every 5 days. Plants were grown in a greenhouse with a day/night mean temperature of 27/20 °C and a relative humidity of 65/80%. Natural light was supplemented with 200 μmol photons m−2 s−1 at the canopy height for 16 h per
Concentration of nitrate, sulfate, reduced N and S compounds
Nitrate concentration was not significantly changed by S-deprivation for the first 3 days, while increased by 2.1-fold in leaves and 1.5-fold in roots (Table 1), respectively, compared to control when prolonged to day 6. S-resupply decreased nitrate concentration to the control level in leaves or 46.9% of control in roots. S-deprivation decreased sulfate concentration both in leaves and roots. Decreased sulfate concentration in the S-deprived plants was recovered to the control level when
Sulfur deprivation treatment induced S-deficient symptom
Following a deprivation of sulfate for 6 days, S-deficiency symptoms occurred as reported previously (Prosser et al., 2001, Koralewska et al., 2009, Honsel et al., 2012). S-deprived plants were characterized by lowered sulfate (Table 1), reduced S compounds (cysteine and glutathione, Fig. 1A, B) and proteins (Fig. 1D), accompanied by enhanced nitrate concentrations in both leaves and roots (Table 1).
Sulfate resupply impacts on the expression of sulfate transporters, sulfate assimilatory enzymes activity and S reduced compounds in S-deprived plants
S-deprivation strongly enhanced expression of Group 1 sulfate transporter (Fig. 2A, B), which
Author contribution
T.H. Kim, B.R. Lee and Q. Zhang designed the experiment. T.H. Kim interpreted data and drafted the manuscript. Q. Zhang carried out the experiment and wrote the manuscript under guidance of Professor T.H. Kim. B.R. Lee, S.H. Park and R. Zaman carried out chemical analysis and statistical analysis. J.C. Avice and A. Ourry participated in data interpretation and critical reading of the manuscript.
Acknowledgments
This study was financially supported by the Korea Research Foundation Grant (NRF-2013R1A2A2A01014202). We gratefully thank Marie-Paule Bataillé for conducting isotopic analysis.
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