Introduction

In plants, free proline accumulation as one of the potential indicators of osmotic tolerance has been correlated with tolerance to drought stresses (Hare and Cress 1997; Ashraf and Foolad 2007), and it also has been thought to play a cardinal role as an osmoregulatory solute subjected to hyperosmotic stresses (Delauney and Vermad 1993). It has been known that there are two alternative routes in proline biosynthesis in higher plants: the l-ornithine and the l-glutamate pathways. And the glutamate pathway is dominant under osmotic stress conditions (Delauney et al. 1993; Kishor et al. 2005). In this pathway, the dual function enzyme Δ1-pyrroline-5-carboxylate synthetase (P5CS) is a key enzyme, which catalyzes the conversion of glutamate into Δ1-pyrroline-5-carboxylate, which is finally reduced to proline. Nitrogen is an extremely important element of plant nutrition; lack of nitrogen commonly limits the growth and development of plant. The proline biosynthesis could also be regulated by the flux of nitrogen by controlling the level of P5CS under stress conditions (Delauney et al. 1993; Kishor et al. 1995).

For the production of plant oils that can be converted into biodiesel, Jatropha curcas is one species that has received much attention recently (Gubitz et al. 1999; Achten et al. 2007; Fairless 2007; King et al. 2009): not only as an energy plant, but also as a drought-tolerant plant used to describe land of poor quality (King et al. 2009). In China, J. curcas has been used to control soil erosion in the Jinsha River dry-hot valley (Li et al. 2007). In this study, we studied the effects of low nitrogen supply and drought stresses on proline synthesis to discuss the tolerance mechanism of J. curcas growing in arid land.

Materials and methods

Plant material, growth conditions, and drought treatment

The seeds of J. curcas were collected from Panxi area in China. After soaking in distilled water for 12 h, the seeds were planted in plastic pot filled with quartz sand and watered with nutrient solution (4 mM N as Ca(NO3)2 and KNO3; 0.5 mM K as KH2PO4; 1.2 mM Mg as MgSO4; 1.0 mM Ca as Ca(NO3)2; 10 μM B as H3BO3; 15 μM Fe as Fe-EDTA; 0.5 μM Mn as MnSO4; 0.5 μM Zn as ZnSO4; 0.5 μM Cu as CuSO4; 0.1 μM Mo as (NH4)6Mo7O24) (Brueck and Senbayram 2009). For low-nitrogen treatments, the N concentrations (KNO3 instead of Ca(NO3)2) were 0.05, 0.20, 0.50, and 1.00 mM (Table 1); potassium and calcium supply of low-nitrogen treatments were balanced by addition of the appropriate amount of KCl and CaCl2. The cultures were in greenhouse at 28 ± 1°C and under 16/8 h (light/dark) photoperiod conditions adjusted to an intensity of 1,000 μmol m−2 s−1.

Table 1 Effects of nitrogen supply on the total biomass and leaf area of Jatropha curcas, 30 days after emergence
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Osmotic treatments were carried out under the same culture conditions. After a growing period of 30 days, the seedlings grown in each nitrogen treatment were exposed to PEG-6000 (−1.6 MPa) stress for 48 h.

Biomass determination and leaf area analysis

The plants were harvested after a growing period of 30 days. The roots were rinsed five times with redistilled water and dried on filter paper. The leaf blades were measured using a portable area meter (CI-202, CID). In a given experimental series, the different plant organs were collected and dried at 105°C for 48 h, then weighed for a dry matter (DW) assessment.

Proline content

Proline content was determined according to the method described by Bates et al. (1973). Fresh leaf material (0.50 g) was homogenized in 10 cm3 of 3% aqueous sulfosalicylic acid and filtered through filter paper. Half milliliter of the filtrate was mixed with 1 ml of acid-ninhydrin and 1 cm3 of glacial acetic acid in a test tube. The mixture was placed in a water bath at 100°C for 1 h. Then, the mixture was extracted with 4 cm3 of toluene and the chromophore containing toluene was aspirated, cooled to room temperature, and the absorbance was measured at 520 nm with a UV/visible spectrophotometer. Appropriate proline standards were included for the calculation of proline in the samples.

RNA isolation and real-time RT–PCR analysis of JcP5CS gene

Total RNA was isolated from J. curcas leaves using RNA prep pure Plant Kit (Tiagen, China) according to the manufacturer’s protocol. Reverse transcription of the pooled RNA was carried out with oligo(dT) primers using ReverTra Ace-α-™ (TOYOBO). The expression of JcP5CS (GeneBank accession No. GU358610) was analyzed quantitatively using SYBR Green II real-time PCR kit (TaKaRa) with Bio-Rad IQ5 detection system. The reaction mixture contained 2 × SYBR Green PCR Master Mix, 18 pM each of the forward and reverse primers of JcP5CS (PR-F: 5′-GGGCAAGCAAGGAGATGAAC-3′, PR-R: 5′-TCCAACCTCAGCACCTAAGC-3′) or 18s rRNA (GeneBank accession No. AY823528) (18sF: 5′-TCGTATCTTGCTGTGACTGG-3′; 18s: 5′-TTGGCAAGCCTTAGCATC-3′), and 1 μl cDNA in a total volume of 25 μl. The thermocycling program was 40 cycles of 95°C for 20 s and 60°C for 20 s with an initial cycle of 95°C for 10 s. After the PCR, a dissociation curve (melting curve) was constructed from the range of 55–95°C to ensure that primer–dimers and other nonspecific products had been eliminated. Data were collected and processed, including baselines subtraction and threshold definition, with iQ5-Cycler software (BioRad).

P5CS activity assay

The extraction of P5CS was processed according to Kishor et al. (1995). The leaves were homogenized in an extraction buffer (100 mM Tris–Cl, pH 7.5, 10 mM β-mercaptoethanol, 10 mM MgCl2, and 1 mM PMSF) in pre-chilled eppendorf tubes on ices. The extracts were centrifuged at 4°C for 20 min at 10,000g. The supernatants were further clarified by centrifugation at 10,000g for 20 min at 4°C. The activity of P5CS was processed according to García-Ríos et al. (1997). The P5CS assay was carried out in 100 mM Tris–Cl (pH 7.2), 25 mM MgCl2, 75 mM Na glutamate, 5 mM ATP, 0.4 mM NADPH, and 50 mg crude extract protein at 25°C. The reaction velocity was measured as the rate of consumption of NADPH, monitored as decrease in absorption at 340 nm as a function of time. The unit of P5CS activity was 0.001 ΔODA340 min−1. Total protein content was determined according to Bradford method (Bradford 1976).

Statistical data analysis

The experiment was conducted in a completely randomized design with 10–15 seedlings per replication. Each treatment was replicated three times. Statistical data analysis was carried out using one-way ANOVAs. Least significant differences (LSD, P ≤ 0.05) were carried out to evaluate the differences in total biomass and leaf area between treatments. All the data were analyzed using Origin software (version 6.00; Microcal Software, Inc.-Northampton, MA 01060 USA).

Results

Effects of low nitrogen supply on growth of J. curcas seedlings

As described in Table 1, the nitrogen supply significantly influenced the growth of J. curcas seedlings. After a growing period of 30 days, the dry biomass and leaf area in the control group could reach about 0.5 g plant−1 and 125 cm2 plant−1, respectively. The lower nitrogen concentrations caused a steady decline in the dry biomass and leaf area. Under the condition of extra-low nitrogen concentration (0.05 mM), the dry biomass decreased to 0.1 g plant−1, which was only one-fifth of the ones in the control group. On the other hand, the leaf area of the seedlings grown in the nutrient solution with 0.05 mM nitrogen was half of the control group.

Effects of low nitrogen and drought stresses on free proline accumulation

Free proline was detected in leaves of J. curcas seedlings grown in nutrient solution with different concentrations of nitrogen with or without PEG stress (Fig. 1). For the treatments without PEG stress, the proline content maintained 25 μg/kg FW under the control and higher nitrogen concentrations (1.00 and 0.50 mM), but when the nitrogen concentrations were at 0.20 and 0.05 mM, the proline content decreased to about 20 μg/kg FW. The proline content in leaves of J. curcas seedlings rose significantly when the PEG-6000 was added; the highest amount measured was found in 0.20 mM nitrogen concentration, in which the proline content was over 50 μg/kg FW. Under the condition of extra-low nitrogen concentration (0.05 mM), the proline content was 45 μg/kg FW, which was still higher than the ones in the control group.

Fig. 1
figure 1

Effects of low nitrogen supply on free proline accumulation of Jatropha curcas with or without PEG stresses

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Effects of low nitrogen and drought stresses on P5CS gene expression and enzyme activity

To determine the effects of low nitrogen supply on the biosynthesis of proline, the mRNA level of JcP5CS gene and P5CS activity were further detected. Low-nitrogen stresses induced JcP5CS gene expression. Regardless of whether the PEG was added, the lowest amounts of the mRNA level of JcP5CS were all observed in the control group, while the highest values were found in the lowest nitrogen concentration (0.05 mM) (Fig. 2). On the other hand, when the nutrient solutions were without PEG-6000, the P5CS activity significantly decreased with the nitrogen concentrations (Fig. 3). However, similar to the free proline accumulation and JcP5CS gene expression, P5CS activity also rapidly increased along with the decline in nitrogen concentration under the PEG stress.

Fig. 2
figure 2

Effects of low nitrogen on JcP5CS gene expression Jatropha curcas with or without PEG stresses

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Fig. 3
figure 3

Effects of low nitrogen supply on P5CS activity Jatropha curcas with or without PEG stresses

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Discussion

Nitrogen supply effects on the biomass have frequently been reported (Wu et al. 2008; Brueck and Senbayram 2009). A low nitrogen supply usually resulted in very low dry matter production (Sheehy Skeffington and Jeffrey 1985). Under the conditions of low nitrogen concentrations, the growth of J. curcas was significantly inhibited. Low nitrogen supply suppressed the biosynthesis of protein, nucleic acid, and other metabolites, eventually resulting in low biomass and leaf area. Several studies have demonstrated that the nitrogen starvation-stress and low-nitrogen stress could induce genes expression in plants (Chen et al. 2003; Fang et al. 2010). The level of P5CS could also be regulated by the nitrogen supply (Delauney et al. 1993; Kishor et al. 1995). In this study, under the conditions of low-nitrogen concentrations, the mRNA levels of JcP5CS gene were higher; however, the P5CS activity and the proline content were significantly less than that of the control group. This indicated that low-nitrogen stress could induce the JcP5CS gene expression, but it also suppressed the P5CS activity and some other metabolites in proline biosynthesis pathway, and eventually led the decline in the content of free proline.

Accumulation of free proline in plant leaves is a commonly observed response to drought stress (Bandurska and Stroiński 2003; Schafleitner et al. 2007). It could act as an osmoregulatory solute to alleviate the damages caused by environmental stresses (Ashraf and Foolad 2007). Although the content of proline in seedlings under the extra-low nitrogen condition was lower than the control group, however, when the J. curcas seedlings grown in the nutrient solution with different nitrogen concentrations were exposed to PEG stresses, the free proline accumulated rapidly, and the content of proline of J. curcas seedlings grown under low-nitrogen conditions were all higher than the control group. The high level of proline contributes to stabilizing sub-cellular structures, scavenging free radicals, and buffering cellular redox potential. Furthermore, it may also function as a protein-compatible hydrotrope (Srinivas and Balasubramanian 1995), alleviating cytoplasmic acidosis, and maintaining appropriate NADP+/NADPH ratios compatible with metabolism (Hare and Cress 1997). That may be one of the reasons why J. curcas could suffer drought stress in arid land.

In plants, the process of proline biosynthesis is orderly and complex, and many factors are involved (Kishor et al. 2005). In this study, the three important factors—content of free proline, mRNA level of JcP5CS gene, and P5CS activity—in proline biosynthesis were detected in J. curcas seedlings under nitrogen and drought stresses. We can clearly find that with the reduction in nitrogen concentration, the counts of all the three factors increased, and among them, the mRNA level of JcP5CS had the highest growth rate, followed by activity of P5CS and the lowest one was the content of free proline. Low-nitrogen stress suppressed some factors in proline biosynthesis pathway, which could result in free proline content decrease. However, when the seedlings stressed by low-nitrogen conditions were exposed to drought stress, the upstream factors of the biosynthesis pathway should be largely synthesized to get sufficient products for the next step. And the ultimate goal of this series of acts was to get enough end-product (proline) to mitigate the impact of drought stress.

In conclusion, our results have shown that low-nitrogen supply suppressed the growth and development of J. curcas seedlings, including the proline biosynthesis. But when the seedlings grown in low-nitrogen conditions were exposed to drought stresses, the JcP5CS gene could be highly induced that led to high P5CS enzyme activity, eventually resulting in proline content’s increase to protect organism from harm. This should be one of the reasons why J. curcas could adapt to poor and drought conditions.