Interaction of sulfate assimilation with carbon and nitrogen metabolism in Lemna minor

Abstract

Cysteine synthesis from sulfide and O-acetyl-L-serine (OAS) is a reaction interconnecting sulfate, nitrogen, and carbon assimilation. Using Lemna minor, we analyzed the effects of omission of CO(2) from the atmosphere and simultaneous application of alternative carbon sources on adenosine 5'-phosphosulfate reductase (APR) and nitrate reductase (NR), the key enzymes of sulfate and nitrate assimilation, respectively. Incubation in air without CO(2) led to severe decrease in APR and NR activities and mRNA levels, but ribulose-1,5-bisphosphate carboxylase/oxygenase was not considerably affected. Simultaneous addition of sucrose (Suc) prevented the reduction in enzyme activities, but not in mRNA levels. OAS, a known regulator of sulfate assimilation, could also attenuate the effect of missing CO(2) on APR, but did not affect NR. When the plants were subjected to normal air after a 24-h pretreatment in air without CO(2), APR and NR activities and mRNA levels recovered within the next 24 h. The addition of Suc and glucose in air without CO(2) also recovered both enzyme activities, with OAS again influenced only APR. (35)SO(4)(2-) feeding showed that treatment in air without CO(2) severely inhibited sulfate uptake and the flux through sulfate assimilation. After a resupply of normal air or the addition of Suc, incorporation of (35)S into proteins and glutathione greatly increased. OAS treatment resulted in high labeling of cysteine; the incorporation of (35)S in proteins and glutathione was much less increased compared with treatment with normal air or Suc. These results corroborate the tight interconnection of sulfate, nitrate, and carbon assimilation.

Figure 1

Effect of incubation in air without CO₂ on APR and NR in L. minor. Liquid cultures of L. minor were cultivated in air without CO₂ (−CO₂) and with the simultaneous addition of 1 mm OAS (−CO₂ + OAS) or 2 mm Suc (−CO₂ + Suc) in continuous light. APR (A), NR (B), and Rubisco (C) activities (o) and mRNA levels (●) were measured at the time points indicated, and Δ indicates enzyme activities in normal air. Mean values ± sd from four measurements of the enzyme activities and mean values of two measurements of mRNA levels are shown. The mRNA level at the beginning of incubation in air without CO₂ was set to 100%.

Figure 2

Effect of the addition of OAS and Suc to plants preincubated in air without CO₂. Liquid cultures of L. minor were cultivated in air without CO₂ for 24 h in continuous light. After reaeration with normal air (+CO₂) or addition of 1 mm OAS (−CO₂ + OAS) and 2 mm Suc (−CO₂ + Suc) and further cultivation in air without CO₂, APR (A) and NR (B) activities (o) and mRNA levels (●) were measured at the time points indicated. Mean values ± sd from four measurements of the enzyme activities and mean values of two measurements of mRNA levels are shown. The mRNA level after 24 h of incubation in air without CO₂ was set to 100%. C, Representative northern blots of APR and NR. Ethidium bromide-stained RNA (RNA) is shown as a control of RNA intactness and loading.

Figure 3

Changes in monosaccharides after reaeration with normal air or the addition of OAS to plants preincubated in air without CO₂. Liquid cultures of L. minor were cultivated in air without CO₂ for 24 h in continuous light. The concentration of Glc and Fru was measured after reaeration with normal air (●) or the addition of 1 mm OAS and further cultivation in air without CO₂ (○) at the time points indicated. █, Indicates control concentrations in normal air. Mean values from two measurements are indicated.

Figure 4

Incorporation of ³⁵S from [³⁵S]sulfate into thiols and proteins. The plants were cultivated in air without CO₂ for 24 h, for 24 h in air without CO₂, for 24 h with normal air, in air without CO₂ for 48 h, and with 1 mm OAS during the last 24 h, and in air without CO₂ for 48 h and with 2 mm Suc during the last 24 h. ³⁵SO₄²⁻ was added to the nutrient solution for the last 4 h of the treatments. Radioactive sulfur (left) and total content (right) of SO₄²⁻, Cys, GSH, and protein was measured. Mean values ± sd of four measurements are presented. Values indicated by different letters represent significant differences at P ≤ 0.01.