Nitrogen (N) and sulphur (S), being essential macronutrients, have important roles in microalgae metabolism. Effects of N- or S-shortage were investigated in the green microalgae Chlorella sorokiniana subjected to 24 h of starvation, by measuring the glutamine synthetase (GS) and O-ace- tylserine(thiol)lyase (OASTL) activities, proteins and amino acids levels. To test possible metabolic impact related to carbon (C) metabolism in response to N- or S-deprivation, starch and total C, N and S contents were also determined. The growth of C. sorokiniana cells was affected by N or S availability. The algae cultured for 24 h in a medium deprived of nitrogen or sulphur showed a decrease in the growth rate and changes in the average volume cell. Nitrogen starvation affected proteins level in the algae cells more than S-deprivation did. The decline in the protein levels observed under S-deficient conditions was coupled with the accumulation of the amide glutamine and with OASTL activity increase; additionally, N-deficiency promoted a decrease in cysteine (Cys) levels (50%) and an increase in GS activity. Nevertheless, S-deprivation had negligible effects on GS activity, while N-deprivation significantly affected OASTL activity. Total C was also estimated in cells N- or S-deprived; nitrogen deprivation strongly affected total C content more than S-deprivation, which in addition reduced the content of C and N, but leaves intact their ratios. Our results support the hypothesis that in Chlorella sorokiniana cells a reciprocal influence of N, S and C assimilation occurs.
Being constituents of important primary metabolites, nitrogen and sulphur represent for all plant cells essential macronutrients, hence their deficiency triggers a wide range of metabolic responses in higher plants and microalgae. To investigate about the effects of mineral nutrients shortage in plant cell, microalgae represent a valuable support for their rapidity of growth, simply culture system and high reproducibility of experiments. Therefore, it is necessary to distinguish the effects due to a long-time starvation from those deriving from a short-time starvation. At this regard it should be pointed out that what for an higher plant is a starvation of short duration in the context of hours, for algae it should be considered a long-time starvation. Metabolic changes associated with nutrient deprivation in the green microalgae Chlorella sorokiniana occurred in a time-dependent manner, generally reaching a maximum in the first day (24 h) of starvation, but depending on the specific growth rate in any organism. In this context, a Chlorella sorokiniana suspension that contained all nutrients in sufficient concentration showed a growth constant of 3 d−1.
In microalgae, long-term nutrient deprivation can lead to the cell death preceded by autophagy, a self-de- grading process to recycle part of the cytoplasm including organelles [
Although nutritional deficiencies determine in algal cells common adaptation strategies, we have previously demonstrated that N- or S-starved cells of Chlorella sorokiniana display different metabolic trends, suggesting that different response mechanisms exist to compensate for the absence of these two nutrients. As a macroelement, N has a profound importance for microalgae metabolism and its limitation is compensated by radical changes in metabolic pathways. N-deprivation stimulates de-repression of enzymes involved in N metabolism such as nitrate reductase [
In this study, the authors determine and compare, in C. sorokiniana, the effects of N- or S-starvation on C metabolism and evaluate the importance of N in the sulphur assimilation and of S in the nitrogen assimilation. In particular, GS and OASTL, key enzymes in N and S assimilation pathways respectively, were taken into consideration. The enzyme GS consents the insertion of inorganic nitrogen into glutamate to form the amide glutamine, while the enzyme OASTL inserts inorganic sulphide into OAS to synthesize cysteine. Many papers indicate these two enzymes are finely regulated in plant cell [
The aim was also to clarify aspects of the interaction among N, S and C metabolisms in a photosynthetically active plant cell. Besides, these findings may contribute to the knowledge of the metabolic consequences to mineral deprivation in microalgae.
All experiments were performed by using Chlorella sorokiniana Shihira & Krauss, strain 211/8K (CCAP of Cambridge University). The algae were grown in batches placed in a thermostatic chamber at 35˚C, continuously stirred and illuminated by fluorescent lamps (150 µmol photons m−2∙s−1). The cultures were insufflated with air containing 5% CO2 at a flow rate of 80 - 100 l∙h−1. Three different types of medium were used to grow cells: basal, nitrate-free and sulphate-free medium. The composition of the basal medium was previously reported [
The packed cell volume (PCV) was estimated by centrifuging 10 ml of cell suspension in a haematocrit tube at 4000 r for 5 min. At the end of centrifugation the PCV value can be directly read from the calibrated tube.
Intracellular glutamine concentration was tested in sufficient and in S- or N-starved cells. Cell suspensions (10 ml) were collected by centrifugation (4000 r for 5 min), the packed cells mixed with a solution containing 1 ml cold 80% ethanol and 1 mM γ-aminobutyric acid, left for 15 min and then centrifuged (4000 r for 5 min). The supernatant was filtered through Waters Sep-Pak® Cartridges to remove chlorophyll. The glutamine was determined by HPLC as previously described [
To prepare crude extract for GS activity determination, aliquots of 100 ml of algal culture were harvested by centrifugation at 4000 r for 5 min; the pellets were re-suspended in 5 ml of extraction buffer ( 10 mM Tris-HCl pH 7.2, 2.5 mM MgCl2, 1 mM dithiothreitol, 5 mM ethylenediaminetetraacetic acid, 15% glycerol) and the cells were lysed by a passage at 11,000 psi through French pressure cell (Aminco). To 1 ml of mix reaction ( 40 mM imidazole-HCl pH 7.0, 30 mM glutamine, 20 mM K-arsenate pH 7.0, 120 mM NH2OH pH 7.0, 0.4 mM adenosine diphosphate, 3 mM MnCl2) 1 ml of crude extract was added. The whole mixture was incubated at 30˚C and after 10 min, 2 ml of stop mixture (4% FeCl3, 2.4% trichloroacetic acid, 0.6 M HCl) was included. Subsequent to a low speed centrifugation the absorbance of the mix was measured at 540 nm. One enzyme U is defined as 1 µmol of γ-glutamylhydroxamate min−1∙mg−1 protein. To determine OASTL activity, aliquots of 100 ml of cultures were harvested by centrifugation (4000 r for 10 min) and the pellets were re-suspended in the extraction buffer ( 50 mM phosphate-buffer pH 7.5, 10 μM pyridoxal phosphate and 1 mM dithiothreitol). The cells were broken by passing twice through a French pressure cell (11,000 psi). The lysates were clarified by centrifugation at 15,000 rpm for 15 min at 4˚C. The supernatant represented the crude extract. The enzymatic activity of OASTL was measured according to Gaitonde method [
Cells were collected by a low speed centrifugation (4000 r for 10 min). The packed cells were extracted twice with 80% ethanol at 80˚C for 15 min. After cooling, the pellets were re-suspended twice in distilled water and then centrifuged. The pellets were washed twice in 50 mM acetic acid-NaOH buffer (pH 4.8) by centrifugation and then autoclaved for 30 min at 120˚C. The extracts were cooled and total starch was determined as glucose derived from its hydrolysis as previously described [
Experimental data analyses were made using Sigmaplot 12 software. All data are expressed as the means ± SE for 5 to 9 determinations. The statistical analysis was performed by one-way ANOVA with a Tukey post-hoc test to determine differences between sufficient and S or N starved algae, P < 0.05 and P < 0.001 as significant. If necessary, the data were log + 1 (x) transformed before the analysis. Data expressed in percentages were trans-
formed by arcsin transformation (y′ = arcsin
In order to define and compare the effects of N- or S-starvation on algae growth, we performed OD550 determination. When cells were in the exponential phase of growth (OD550 about 0.8), they were starved of N- or S-nu- trient and the growth monitored for the following 48 h of nutrient deprivation (
Some previous papers reported reduction in microalgal growth rate and increase of cell volume as a consequence of long-term N- and S-starvation [
We observed that cellular growth is more affected in N-starved cells if compared to S-starved ones (
S-deprivation implies a reduction of cell growth that is less evident than in N-starvation: the fact that in S- starved cells the rate of growth has a slowdown delayed probably corresponds to a period of cellular sulphur recycling. As previously reported in Chlorella sorokiniana cells [
Our studies showed that N-starvation generally yielded similar effects as S-starvation, but the impacts on cell growth and total protein were much more severe.
Culture condition | Starch mg glucose mL−1 PCV |
---|---|
Sufficient | 82.6 ± 3.1 |
N-starved | 256.5 ± 6.7 |
S-starved | 201.3 ± 9.4 |
Total soluble protein concentration in algal samples decreased drastically in response to nutrient starvation. A 65% decrease in protein content was observed after 24 h of S-deprivation and a decrease of over 80% was observed after 24 h of N-starvation (
Strikingly, S-deficiency also affects free amino acids pool, which appeared greatly increased compared to sufficient algae [
N- or S-deprivation causes a loss of photosynthetic capacity and a decrease in the cells chlorophyll content. As previously reported, chlorophyll content strongly decreased upon both S- and N-starvation [
bisphosphate carboxylase oxygenase [
Our previous results [
Our recent investigation showed that activation of antioxidant enzymes in S-deprived C. sorokiniana occurred in a period of 24 h of S-starvation and that these increases correspond to the rapid raise in Reactive Oxygen Species (ROS) occurring in the first hour of starvation [
In this paper we show that total C is reduced by 10% in N-deprived and 16% in S-deprived cells. Another interesting result is that in cells S-deprived the total N is reduced by only 23%, whereas in cells N-deprived the total S is reduced by 47%. These data seem to confirm that N-deprivation strongly affects S-assimilation (
Culture condition | Glutamine synthetase (GS) U mg∙prot−1 | O-acetylserine(thiol)lyase (OASTL) U mg∙prot−1 |
---|---|---|
Sufficient | 2.1 ± 0.4 | 3.1 ± 0.28 |
N-starved | 4.3 ± 0.5 | 0.3 ± 0.04 |
S-starved | 2.4 ± 0.5 | 15.8 ± 1.62 |
It has also been shown that N-deprivation increases the C/N ratio (6.4), probably due to reduced synthesis of amino acids and proteins and to the storage of starch. In S-starved cells the C/N ratio slightly increased (4.7) respect to that of sufficient cells (4.3).
N- and S-deprivation influence C metabolism as photosynthetic activity and starch storing in a similar mode. Anyway, cellular growth is more affected in N-starved cells if compared to S-starved ones. S-deprivation implies a reduction of cell growth but with delay with respect to the N-starvation. Probably, the cellular recycling in response to nutrient deprivation is more effective under S-respect to N-shortage. In addition, the relative importance and abundance of N compared with S could explain this growth discrepancy between N- and S-starved cells. The total N content in C. sorokiniana cells is estimated to be almost 10-fold greater than the total S content. Our data show that N-deprivation powerfully affects S-assimilation. In N-deprived cells the total S is reduced by 47%, whereas in S-deprived cells the total N is reduced by 23%. The cysteine intracellular content decreased in N-starved cells by around 50% and OASTL activity resulted strongly reduced. Our data demonstrate that in Chlorella sorokiniana a mutual influence of N, S and C assimilation occurs.