Nitrogen-sufficient Conditions Research Articles

Lipid Production by Arctic Microalga Chlamydomonas sp. KNF0008 at Low Temperatures.

A lipid-producing microalga, Chlamydomonas sp. KNF0008, collected from the Arctic was capable of growing at temperatures ranging from 4 to 20°C, and the highest cell density was measured at 15°C and 100μmol photonsm-2s-1 light intensity under continuous shaking and external aeration. KNF0008 showed the elevated accumulation of lipid bodies under nitrogen-deficient conditions, rather than under nitrogen-sufficient conditions. Fatty acid production of KNF0008 was 4.2-fold (104mgL-1) higher than that of C. reinhardtii CC-125 at 15°C in Bold's Basal Medium. The dominant fatty acids were C16:0, C16:4, C18:1, and C18:3, and unsaturated fatty acids (65.69%) were higher than saturated fatty acids (13.65%) at 15°C. These results suggested that Arctic Chlamydomonas sp. KNF0008 could possibly be utilized for production of biodiesel during periods of cold weather because of its psychrophilic characteristics.

Nitrogen Supply and Leaf Age Affect the Expression of TaGS1 or TaGS2 Driven by a Constitutive Promoter in Transgenic Tobacco.

Glutamine synthetase (GS) plays a key role in nitrogen metabolism. Here, two types of tobacco transformants, overexpressing Triticum aestivum GS1 (TaGS1) or GS2 (TaGS2), were analysed. Four independent transformed lines, GS1-TR1, GS1-TR2, GS2-TR1 and GS2-TR2, were used for the nitrogen treatment. Under nitrogen-sufficient conditions, the leaves of GS2-TR showed high accumulation of the TaGS2 transcript, while those of GS1-TR showed a low TaGS1 transcript levels. However, compared with nitrogen-sufficient conditions, the TaGS1 transcript level increased in the leaves under nitrogen starvation, but the TaGS2 transcript level decreased. In addition, the TaGS1 and TaGS2 transcript levels were highest in the middle leaves under nitrogen-sufficient and starvation conditions. These results show that nitrogen supply and leaf age regulate TaGS expression, even when they are driven by a super-promoter. Additionally, in regard to nitrogen metabolism level, the lower leaves of the GS1-TR exhibited lower NH4+ and higher amino acid contents, while the upper leaves exhibited higher amino acid, soluble protein and chlorophyll contents. The leaves of the GS2-TR exhibited lower NH4+ but higher amino acid, soluble protein and chlorophyll contents. Given the role that GS isoforms play in nitrogen metabolism, these data suggest that TaGS1 overexpression may improve nitrogen transport, and that TaGS2 overexpression may improve nitrogen assimilation under nitrogen stress.

Enhanced Production of Fatty Acids via Redirection of Carbon Flux in Marine Microalga Tetraselmis sp.

Lipids in microalgae are energy-rich compounds and considered as an attractive feedstock for biodiesel production. To redirect carbon flux from competing pathways to the fatty acid synthesis pathway of Tetraselmis sp., we used three types of chemical inhibitors that can block the starch synthesis pathway or photorespiration, under nitrogen-sufficient and nitrogen-deficient conditions. The starch synthesis pathway in chloroplasts and the cytosol can be inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and 1,2-cyclohexane diamine tetraacetic acid (CDTA), respectively. Degradation of glycine into ammonia during photorespiration was blocked by aminooxyacetate (AOA) to maintain biomass concentration. Inhibition of starch synthesis pathways in the cytosol by CDTA increased fatty acid productivity by 27% under nitrogen deficiency, whereas the blocking of photorespiration in mitochondria by AOA was increased by 35% under nitrogen-sufficient conditions. The results of this study indicate that blocking starch or photorespiration pathways may redirect the carbon flux to fatty acid synthesis.

Homologous expression of lipid droplet protein-enhanced neutral lipid accumulation in the marine diatom Phaeodactylum tricornutum

Lipid droplets are ubiquitous cellular compartments that store neutral lipids and specific proteins localize on their surface. These proteins work as a scaffold in maintaining the lipid droplet structure or as regulators of lipogenesis or lipolysis. Previously, the most abundant lipid droplet protein, namely stramenopile-type lipid droplet protein (StLDP), was identified in the marine diatom Phaeodactylum tricornutum; however, its function remains unclear because StLDP does not reveal homology with known lipid droplet proteins and lacks a predictable domain. In this study, P. tricornutum was transformed to express a homologous StLDP gene under an fcpA promoter in order to determine its function. StLDP expression was strongly enhanced in the mutant (H8), especially in nitrogen-sufficient conditions; however, it was attenuated in nitrogen-deficient conditions. Despite the strong expression, no significant difference was observed in the lipid composition between the wild type (WT) and H8 under nitrogen-sufficient conditions. After cultivation in nitrogen-free medium for 6 days, neutral lipid content significantly increased in H8 than in WT. After 2 days of cultivation in nitrogen-free medium, 97.0% of single cells in WT formed one or two lipid droplets, whereas in H8, this proportion decreased to 78.8%, and the proportion of cells forming three or four lipid droplets increased. Thus, the function of StLDP was speculated to sequester triacylglycerol on the initial lipid droplet formation.

Excessive phosphorus enhances Chlorella regularis lipid production under nitrogen starvation stress during glucose heterotrophic cultivation

Recent studies claimed nitrogen starvation in addition to sufficient phosphorus content to be a “lipid trigger” for microalgae autotrophic cultivation. However, the role of phosphorus in heterotrophic cultivation still remains controversial. Here, we investigated the utilization of a series of phosphorus concentrations of up to 818% in BG11 medium to cultivate Chlorella regularis under nitrogen starvation stress during glucose heterotrophic cultivation. Phosphorus positively influenced microalgal cell synthesis, and the lipid content increased with increasing levels of supplied phosphorus. The excess phosphorus was stored in the form of Poly-P, thus providing energy and participating in the synthesis of cellular materials, which enhanced cell growth and lipid accumulation. The biomass production reached 4.53g/L under nitrogen starvation with excess phosphorus supply, which was comparable to the effect of a nitrogen-sufficient condition (control). The excessive phosphorus level upregulated the genes ROT3, BAS1, CYP735A, FBP, GOT2, and AOC3 associated with growth. Microalgal lipid content reached 42.3% and lipid productivity reached 310.0mg/(L·d), values that were 2 and 1.3times higher than those in the control, respectively. Furthermore, the additional phosphorus supply increased the amounts of saturated fatty acids, while shifting fatty acid pathways. We suggest that higher amounts of phosphorus should be supplied to achieve high microalgal lipid productivity under nitrogen starvation stress during glucose heterotrophic cultivation. This is an effective strategy to enhance biodiesel production, simultaneously achieving high biomass production and efficient lipid accumulation.

The chimeric repressor for the GATA4 transcription factor improves tolerance to nitrogen deficiency in Arabidopsis.

Nitrogen limits crop yield, but application of nitrogen fertilizer can cause environmental problems and much fertilizer is lost without being absorbed by plants. Increasing nitrogen use efficiency in plants may help overcome these problems and is, therefore, an important and active subject of agricultural research. Here, we report that the expression of the chimeric repressor for the GATA4 transcription factor (35S:GATA4-SRDX) improved tolerance to nitrogen deficiency in Arabidopsis thaliana. 35S:GATA4-SRDX seedlings were significantly larger than wild type under nitrogen-sufficient and -deficient conditions (10 and 0.5 mM NH4NO3, respectively). 35S:GATA4-SRDX plants exhibited shorter primary roots, fewer lateral roots, and higher root hair density compared with wild type. The expression levels of NITRATE TRANSPORTER 2.1, ASPARAGINE SYNTHETASE and NITRATE REDUCTASE 1 were significantly higher in roots of 35S:GATA4-SRDX plants than in wild type under nitrogen-sufficient conditions. Under nitrogen-deficient conditions, the expression of genes for cytosolic glutamine synthetases was upregulated in shoots of 35S:GATA4-SRDX plants compared with wild type. This upregulation of nitrogen transporter and nitrogen assimilation-related genes might confer tolerance to nitrogen deficiency in 35S:GATA4-SRDX plants.

Effects of nitrogen availability on polymalic acid biosynthesis in the yeast-like fungus Aureobasidium pullulans.

BackgroundPolymalic acid (PMA) is a novel polyester polymer that has been broadly used in the medical and food industries. Its monomer, L-malic acid, is also a potential C4 platform chemical. However, little is known about the mechanism of PMA biosynthesis in the yeast-like fungus, Aureobasidium pullulans. In this study, the effects of different nitrogen concentration on cell growth and PMA biosynthesis were investigated via comparative transcriptomics and proteomics analyses, and a related signaling pathway was also evaluated.ResultsA high final PMA titer of 44.00 ± 3.65 g/L (49.9 ± 4.14 g/L of malic acid after hydrolysis) was achieved in a 5-L fermentor under low nitrogen concentration (2 g/L of NH4NO3), which was 18.3 % higher yield than that obtained under high nitrogen concentration (10 g/L of NH4NO3). Comparative transcriptomics profiling revealed that a set of genes, related to the ribosome, ribosome biogenesis, proteasome, and nitrogen metabolism, were significantly up- or down-regulated under nitrogen sufficient conditions, which could be regulated by the TOR signaling pathway. Fourteen protein spots were identified via proteomics analysis, and were found to be associated with cell division and growth, energy metabolism, and the glycolytic pathway. qRT-PCR further confirmed that the expression levels of key genes involved in the PMA biosynthetic pathway (GLK, CS, FUM, DAT, and MCL) and the TOR signaling pathway (GS, TOR1, Tap42, and Gat1) were upregulated due to nitrogen limitation. Under rapamycin stress, PMA biosynthesis was obviously inhibited in a dose-dependent manner, and the transcription levels of TOR1, MCL, and DAT were also downregulated.ConclusionsThe level of nitrogen could regulate cell growth and PMA biosynthesis. Low concentration of nitrogen was beneficial for PMA biosynthesis, which could upregulate the expression of key genes involved in the PMA biosynthesis pathway. Cell growth and PMA biosynthesis might be mediated by the TOR signaling pathway in response to nitrogen. This study will help us to deeply understand the molecular mechanisms of PMA biosynthesis, and to develop an effective process for the production of PMA and malic acid chemicals.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0547-y) contains supplementary material, which is available to authorized users.

Regulatory mechanisms of oxidative species and phytohormones in marine microalgae Isochrysis zhangjiangensis under nitrogen deficiency

Oxidative species and phytohormones are important factors in plants that can regulate stress responses. In this study, the content variations of four oxidative species, O2−, H2O2, ClO−, and NO, and the phytohormones, salicylic acid (SA), jasmonic acid (JA), and folic acid (FA), were investigated in the microalgae Isochrysis zhangjiangensis under nitrogen deficiency. The O2− and H2O2 contents were found to be positively correlated with the photosynthetic activity of the microalgae, while the opposite relationship was observed for NO and ClO−. In higher plants, there is a balance model for NO and ROS interactions, which revealed that the cooperation of NO and H2O2 regulates the pathogen-induced hypersensitive response-associated cell death. Because the NO and H2O2 levels in both nitrogen-replete and nitrogen-depleted I. zhangjiangensis were not simultaneously high, we proposed that as a unicellular lower plant, the microalgae I. zhangjiangensis follows this model and regulates the different types of intracellular oxidative species, especially when they were cultured from nitrogen sufficient condition to corresponding nitrogen deficient condition, to avoid cell death. Together with the corresponding changes in the SA, JA and FA contents in the nitrogen-stressed microalgae, we proposed that the balance model of oxidative species interactions, as well as the correlations among nitrogen assimilation, photosynthesis and the biosyntheses of SA, JA, and FA, may originally exist in algae, and plants have followed this model and maintained these correlations all along to protect themselves from stressful conditions during their evolutions from alga to higher plants.

Combining nitrogen starvation with sufficient phosphorus supply for enhanced biodiesel productivity of Chlorella vulgaris fed on acetate

Heterotrophic cultivation of microalgae is attracting more attention due to its high production efficiency. In this study, Chlorella vulgaris was cultivated heterotrophically with acetate in batch systems. The effects of nitrogen deficiency and different phosphorus supply levels on biodiesel production were investigated. It was found that C. vulgaris can assimilate acetate and accumulate fatty acids simultaneously. The highest FAME (Fatty acid methyl ester) content (56%) of algae was obtained in nitrogen deficient media. Interestingly, the productivity of fatty acids under nitrogen starved conditions was three times greater than that under nitrogen sufficient conditions. Moreover, FAME productivity was further enhanced to 66mgL−1·d−1 by a sufficient phosphorus supply but there was no effect caused by phosphorus limitation. Furthermore, the conversion yields of acetate consumed to fatty acids produced (Chemical oxygen demand (COD)-based) in nitrogen starved media were also three times higher than those in nitrogen sufficient media. FAME productivity of C. vulgaris under nitrogen starvation was not improved by controlling the pH at stable values (7.5, 8.0, and 8.5), and even decreased when pH was controlled at 7.0 and 9.0.

Structural basis for Mep2 ammonium transceptor activation by phosphorylation.

Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear. Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation.

Correlating Gene Expression Profiles to Lipid Induction in the Microalga Chlorella protothecoides

Microalgae are a promising source for sustainable biofuel production, but large‐scale production can be cost‐prohibitive. An understanding of the relationship between algal genome and metabolic pathways could serve to decrease cost and increase commercial viability. In this study, we examine the relationship between gene expression and lipid induction in Chlorella protothecoides. Using NextGen sequencing, we identified candidate genes, which were differentially expressed in algae grown in nitrogen sufficient (low lipid production) and nitrogen deficient (high lipid production) conditions. Using TaqMan real‐time PCR, differential expression was confirmed and quantified for each candidate gene in algae grown in media of varying nitrogen concentrations, and patterns of expression were defined. Systematic alterations were then made in pH, light, and micronutrients, and new expression profiles were generated. These expression profiles were correlated with cellular concentrations of lipids measured by lipid stain and flow cytometry to establish a distinct gene expression profile for lipid producing C. protothecoides. These expression profiles provide insight into C. protothecoides specific lipid production pathways. In addition, these lipid induction profiles are currently being used to rapidly assess the impact of environmental factors on lipid production days before detectable lipid production would be quantified.Support or Funding InformationResearch Scholars Program, Kentucky NSF EPScOR

The role of light and nitrogen in growth and carotenoid accumulation in Scenedesmus sp.

To study the regulation of carotenoid biosynthesis and accumulation, the microalga Scenedesmus sp. might represent an alternative model microorganism to the well established Haematococcus pluvialis or Dunaliella salina. Scenedesmus sp. strain CCALA 1074 can produce high levels of carotenoids, mainly lutein, over all growth phases. In exponentially growing cultures, total carotenoids and lutein accumulated to levels of up to 1 and 0.75% DW, respectively. In early stationary phase, total carotenoids increased to 2.3% DW; lutein however remained unchanged and additional minor ketocarotenoids such as astaxanthin, echinenone and a number of unidentified carotenoid species were also present. Nitrate-limited cultures were supplemented either with nitrate or complete growth medium in early stationary phase and were cultivated either in light or in darkness. As expected, in light, cells started to divide and cultures grew; surprisingly however, in darkness, cell division was blocked after supplementation, independent of the nutrient added. Carotenoid synthesis also only proceeded in the light, whereas, in darkness, carotenoid content remained constant. When cultivated in an outdoor thin-layer photobioreactor (PBR), the greatest increase in carotenoid accumulation occurred under conditions of nitrogen sufficiency and high light. These results suggest that in Scenedesmus sp., light is a critical factor in the accumulation of carotenoids, and nitrogen availability plays only a minor role.

Enhancement of FAME productivity of Scenedesmus obliquus by combining nitrogen deficiency with sufficient phosphorus supply in heterotrophic cultivation

In this study, Scenedesmus obliquus NIES-2280 was cultivated heterotrophically with acetate as the carbon source. The effects of nitrogen deficiency and different phosphorus supply levels on biodiesel production by S. obliquus were investigated. It was found that S. obliquus could make good use of assimilated acetate for fatty acid accumulation. Fatty acid contents of algae in nitrogen deficiency media increased to 38–48% after 6-day cultivation. Interestingly, the productivity of fatty acid methyl esters (FAMEs) under nitrogen starved conditions increased fourfold than that under nitrogen sufficient conditions. Moreover, FAME productivity could be further enhanced by a sufficient phosphorus supply rather than under P limitation or P deficiency conditions, and the highest FAME productivity was 55.9mgL−1d−1. Furthermore, the conversion yields of acetate to fatty acids (COD based) in nitrogen starvation media (18–28%) were much higher than those in nitrogen sufficient media (∼7%). This study indicates a great potential to combine wastewater treatment with biodiesel production via S. obliquus which can significantly improve biodiesel productivity and COD utilization under nitrogen starvation coupled with sufficient phosphorus supply.

High temperature enhances lipid accumulation in nitrogen-deprived Scenedesmus obtusus XJ-15

This study investigated the changes in lipid and starch contents, lipid fraction, and lipid profile in the nitrogen-starved Scenedesmus obtusus XJ-15 at different temperatures (17, 25, and 33 A degrees C). The optimal temperature for both growth and lipid accumulation under nitrogen-sufficient condition was found to be 25 A degrees C. However, under nitrogen deprivation, the total and neutral lipids increased with increasing temperature, and achieved the highest lipid content of 47.60 % of dry cell weight and the highest TAG content of 79.66 % of total lipid at 33 A degrees C. In the meantime, the stored cellular starch content decreased with the increasing temperature. Thus, high temperature induced carbon flux from starch toward TAG accumulation in microalgae during nitrogen starvation. In addition, the decreased polar lipids may also serve for TAG synthesis under high temperature, and high temperature further reduced the degree of the fatty acid unsaturation and favored a better biodiesel production. These results suggested that high-temperature stress can be a good strategy for enhancing biofuel production in oleaginous microalgae during nitrogen deficiency.

Biosynthesis of high yield fatty acids from Chlorella vulgaris NIES-227 under nitrogen starvation stress during heterotrophic cultivation

In this study the heterotrophic cultivation of Chlorella vulgaris NIES-227 fed with glucose was investigated systematically using six media types; combinations of nitrogen repletion/depletion and phosphorus repletion/limitation/depletion. It was found that a high yield of fatty acids (0.88 of fed glucose-COD) and a high content of fatty acid methyl esters (FAMEs) (89% of dry weight) were obtained under nitrogen starved conditions. To our knowledge it is the first report on such high COD conversion yield and FAME content in microalgae. The dominant fatty acid (>50%) was methyl oleate (C18:1), a desirable component for biodiesel synthesis. FAME content under nitrogen starved conditions was significantly higher than under nitrogen sufficient conditions, while phosphorus had no significant influence, indicating that nitrogen starvation was the real “fatty acids trigger” in heterotrophic cultivation. These findings could simplify the downstream extraction process, such as the extrusion of oil from soybeans, and could reduce operating costs by improving the fatty acid yield from waste COD.

HY5 regulates nitrite reductase 1 (NIR1) and ammonium transporter1;2 (AMT1;2) in Arabidopsis seedlings

HY5 (Long Hypocotyles 5) is a key transcription factor in Arabidopsis thaliana that has a pivotal role in seedling development. Soil nitrogen is an essential macronutrient, and its uptake, assimilation and metabolism are influenced by nutrient availability and by lights. To understand the role of HY5 in nitrogen assimilation pathways, we examined the phenotype as well as the expression of selected nitrogen assimilation-related genes in hy5 mutant grown under various nitrogen limiting and nitrogen sufficient conditions, or different light conditions. We report that HY5 positively regulates nitrite reductase gene NIR1 and negatively regulates the ammonium transporter gene AMT1;2 under all nitrogen and light conditions tested, while it affects several other genes in a nitrogen supply-dependent manner. HY5 is not required for light induction of NIR1, AMT1;2 and NIA genes, but it is necessary for high level expression of NIR1 and NIA under optimal nutrient and light conditions. In addition, nitrogen deficiency exacerbates the abnormal root system of hy5. Together, our results suggest that HY5 exhibits the growth-promoting activity only when sufficient nutrients, including lights, are provided, and that HY5 has a complex involvement in nitrogen acquisition and metabolism in Arabidopsis seedlings.

Robust biological nitrogen fixation in a model grass-bacterial association.

Nitrogen-fixing rhizobacteria can promote plant growth; however, it is controversial whether biological nitrogen fixation (BNF) from associative interaction contributes to growth promotion. The roots of Setaria viridis, a model C4 grass, were effectively colonized by bacterial inoculants resulting in a significant enhancement of growth. Nitrogen-13 tracer studies provided direct evidence for tracer uptake by the host plant and incorporation into protein. Indeed, plants showed robust growth under nitrogen-limiting conditions when inoculated with an ammonium-excreting strain of Azospirillum brasilense. (11)C-labeling experiments showed that patterns in central carbon metabolism and resource allocation exhibited by nitrogen-starved plants were largely reversed by bacterial inoculation, such that they resembled plants grown under nitrogen-sufficient conditions. Adoption of S. viridis as a model should promote research into the mechanisms of associative nitrogen fixation with the ultimate goal of greater adoption of BNF for sustainable crop production.

Humic substances stimulate maize nitrogen assimilation and amino acid metabolism at physiological and molecular level

Abstract Background The effects of a humic substance (HS) extracted from a volcanic soil on the nitrate assimilation pathway of Zea mays seedlings were thoroughly examined using physiological and molecular approaches. Plant growth, the amount of soluble proteins and amino acids, as well as the activities of the enzymes involved in nitrogen metabolism and Krebs cycle, were evaluated in response to different HS concentrations (0, 1 and 5 mg C L−1) supplied to maize seedlings for 48 h. To better understand the HS action, the transcript accumulation of selected genes encoding enzymes involved in nitrogen assimilation and Krebs cycle was additionally evaluated in seedlings grown for 2 weeks under nitrogen (N) sufficient condition and N deprivation. Results HS at low concentration (1 mg C L−1) positively influenced nitrate metabolism by increasing the content of soluble protein and amino acids synthesis. Furthermore, the activity and transcription of enzymes functioning in N assimilation and Krebs were significantly stimulated. Conclusions HS treatment influenced the gene expression of Zea mays plants at transcriptional level and this regulation was closely dependent on the availability of nitrate in the growth medium.

Fed-batch strategy for enhancing cell growth and C-phycocyanin production of Arthrospira (Spirulina) platensis under phototrophic cultivation

The C-phycocyanin generated in blue-green algae Arthrospira platensis is gaining commercial interest due to its nutrition and healthcare value. In this study, the light intensity and initial biomass concentration were manipulated to improve cell growth and C-phycocyanin production of A.platensis in batch cultivation. The results show that low light intensity and high initial biomass concentration led to increased C-phycocyanin accumulation. The best C-phycocyanin productivity occurred when light intensity and initial biomass concentration were 300μmol/m2/s and 0.24g/L, respectively. The fed-batch cultivation proved to be an effective strategy to further enhance C-phycocyanin production of A.platensis. The results indicate that C-phycocyanin accumulation not only requires nitrogen-sufficient condition, but also needs other nutrients. The highest C-phycocyanin content (16.1%), production (1034mg/L) and productivity (94.8mg/L/d) were obtained when using fed-batch strategy with 5mM medium feeding.

In vitro method for early evaluation of nitrogen use efficiency associated traits in potato

The objective of the present study was to characterize various traits associated with nitrogen uptake and utilization in a range of potato cultivars. For this purpose an in vitro test system was developed which allows analyzing specific stress responses in a highly controlled environment. Shoot tips were grown fixed in perforated stainless steel plates in 500 ml glass vessels in liquid culture medium at four nitrogen levels, i.e. 60, 30, 15 and 7.5 mmol L-1. At the end of a three weeks’ culture period plant developmental traits were determined and nitrogen uptake and assimilation were analyzed. Reduction of nitrogen in the culture medium differentially affected morphological and physiological features. Highly significant differences were found between different N-levels and cultivars as well as for genotype x nitrogen level interactions. Three groups of cultivars (high, low and intermediate) were distinguished with respect to biomass production and crude protein yield under nitrogen-sufficient conditions of 60 mmol L-1. Genotypes with a low biomass production at full nitrogen availability responded with increased root development under nitrogen deficiency stress and increased their nitrogen utilization capacity in relation to the other cultivars.