INTRODUCTION
Rapeseed (Brassica napus var. oleifera Del.) is one of the most important oil crops all over the world and the most important in Europe for both food and non-food (fuel, lubricant) purposes (FAO, 2002). Rapeseed is also widely cultivated for production of solid biomass, forage and for green manuring and has potential for phytoremediation of soils contaminated with toxic metals, organics and radionuclides (Pradhan et al., Reference Pradhan, Conrad, Paterek and Srivastava1998).
The species is important in Mediterranean countries like Spain, Greece and Italy, with regions characterised by semi-arid climates, where the crop can be cultivated in autumn–spring without irrigation and it represents one of the few alternatives for rotation with winter cereals. In these countries, in order to allow the crop to develop enough to tolerate winter frost, an early sowing is needed (late summer–early autumn) and this implies that germination and initial growth can occur under water deficiency (Anastasi et al., Reference Anastasi, Santonoceto and Monti2003). In coastal regions, soils may also have high salinity, which may hamper seed germination and early seedling growth (Ruan et al., Reference Ruan, Teixeira da Silva, Mopper, Qin and Lutts2010). Although rapid and uniform seed germination and early seedling establishment are critical aspects for crop production under saline and drought conditions, a number of studies have established that both seed germination and seedling emergence in canola (Brassica rapa, B. napus) are negatively affected by low soil water potentials and salinity (Al-Thabet et al., Reference Al-Thabet, Leilah and Al-Hawass2004; Andalibi et al., Reference Andalibi, Zangani and Nazari2005; Blackshaw, Reference Blackshaw1991; Willenborg et al., Reference Willenborg, Gulden, Johnson and Shirtliffe2004).
Salt and water stresses may induce numerous metabolic damages in seeds, such as modification of enzyme activities (Ashraf et al., Reference Ashraf, Afaf, Qureshi, Sarwar and Naqvi2002), excessive uptake of ions and reduced mobility of inorganic nutrients to developing tissue (Murillo-Amador et al., Reference Murillo-Amador, Lopez-Aguilar, Kaya, Larrinaga-Mayoral and Flores-Hernandez2002), disturbance in nitrogen metabolism (Dell'Aquila and Spada, Reference Dell'Aquila and Spada1993), imbalances in the levels of plant growth regulators (Khan and Rizvi, Reference Khan and Rizvi1994) and reduction in hydrolysis and utilisation of food reserves (Lin and Kao, Reference Lin and Kao1995). Accumulation of compatible osmotica, such as soluble sugars and free proline, allows osmotic adjustment of stressed tissues to cope with these environmental constraints, although such a process also has a metabolic cost that may affect the kinetics of seed germination (Ashraf et al., Reference Ashraf, Zafar and Ashraf2003).
In rapeseed, it is not clear yet whether gene expression, protein synthesis and cell division are required to allow seed germination to proceed, and whether external constraints, such as water or salt stress, act on these crucial steps. The use of specific inhibitors, such as actinomycin-D (Act-D: a transcription inhibitor), cycloheximide (Cx: inhibitor of translation), hydroxyurea (Hx: inhibitor of DNA synthesis) and cytochalasine (CD: inhibitor of actin polymerisation), could afford valuable information on the nature of the key events involved in germination (Li et al., Reference Li, Feng, Han and Zhu2006; Rajjou et al., Reference Rajjou, Gallardo, Debeaujon, Vandekerckhove, Job and Job2004).
Improving seed germination is an important prerequisite for cultivation of rapeseed in the presence of salt or water stress. Seed vigour can be enhanced by priming treatments that consist of imbibing dry seeds with specific chemicals for given periods, followed by washing and re-drying the seeds to their original moisture concentration or direct seeding of primed material (Harris, Reference Harris2006). Positive effects of priming were observed in oilseed species such as sunflower (Kaya et al., Reference Kaya, Okc, Atak, Cıkılı and Kolsarıcı2006) but few data are available for rapeseed (Afzal et al., Reference Afzal, Aslam, Hameed and Ahmad2008). There are several indications that many physiological mechanisms are involved in the increased vigour of primed seeds such as the repair of the age-related cellular and subcellular damage that accumulates during seed development (Bray et al., Reference Bray, Davision, Ashraf and Taylor1989), advancement of metabolic events during the prolonged lag phase that precedes radicle protrusion (Dell'Aquila and Bewley, Reference Dell'Aquila and Bewley1989) or partial hydrolysis of endosperm materials that allows faster radicle elongation and seedling growth (Burgass and Powell, Reference Burgass and Powell1984; de Lespinay et al., Reference de Lespinay, Lequeux, Lambillotte and Lutts2010). The choice of an efficient priming strategy requires a better understanding of the biochemical processes sustaining seed germination in rapeseed.
The present study was carried out in order to (i) examine the effects of increasing salt stress and matric stress induced by polyethylene glycol on germination in tolerant and sensitive rapeseed cultivars; (ii) determine the impact of metabolic inhibitors on seed germination and (iii) test different priming techniques to improve seed germination under stressing conditions.
MATERIAL AND METHODS
Study of germination under salt and matric stresses
Seeds of rapeseed cultivars Exagone (Monsanto) and Toccata (Maisadour Semences), which have been found to be tolerant and sensitive to salt and water stress, respectively (Pace and Benincasa, Reference Pace and Benincasa2010) and have similar imbibition kinetics when incubated in distilled water, were incubated in PEG (PEG 6000; matric potentials of −0.03, −0.15, −0.3, −0.4, −0.5, −0.6, −0.7, −0.8, −0.9, −1, −1.1, −1.2 and −1.5 MPa) or NaCl (50, 100, 150, 200, 250, 275, 300, 325, 350, 400, 500 and 600 mM corresponding, respectively to an external osmotic potential of −0.23, −0.42, −0.71, −0.92, −1.15, −1.32, −1.41, −1.52, −1.74, −2.03 and −2.21 MPa, assessed by the use of a Wescor vapour pressure osmometer) and in distilled water (control). Matric potentials for PEG concentrations were calculated according to Michel and Kaufmann (Reference Michel and Kaufmann1973). For each of the treatments, three replicates of 50 seeds were incubated in absorbent paper (filter paper circles, 8.5 cm diameter, Whatman Grade 1) on Petri dishes placed in darkness at 20 °C. The number of germinated seeds was counted every day until 14 days from the start of the test. A seed was considered as germinated when radicle was longer than 2 mm.
Seed germination in the presence of metabolic inhibitors
For each of the two cultivars, seeds were incubated in distilled water (control), PEG 6000 (−0.9 MPa) or NaCl (350 mM) solutions containing 100 μM actinomycin-D (Act-D), cycloheximide (Cx), hydroxyurea (Hx) or cytochalasine (CD) (all chemicals purchased by Sigma Aldrich Belgium). Dishes (three dishes, each containing 50 seeds per treatment) were placed in darkness at 20 °C. The number of germinated seeds was counted every day until 8 days from the start of the test.
Results were expressed as final percent germination (G) and time to reach 50% G (T50) (Coolbear et al., Reference Coolbear, Francis and Grierson1984) according to the following equation:
where N is the final number of germinated seeds, and ni and nj are the total number of seeds germinated at times ti and tj, where ni < (N + 1)/2 < nj. For each of the two cultivars, moisture concentration was recorded at 0 (dry seeds), 2, 4, 6, 8, 10, 24, 36, 48, 60 and 72 h after the start of imbibition; seeds were removed from the dish, blotted, weighed and then returned to the wet filter paper. Based on previous evidences on Trifolium repens and Festuca rubra (de Lespinay, Reference de Lespinay2009), the temporary surface-drying of seeds was assumed not to affect the germination process. The water uptake was expressed as the percentage increase in moisture concentration on fresh weight basis.
Priming treatments
For each cultivar, six treatments (four batches of 50 seeds per treatment) were applied: (a) no priming; priming in; (b) distilled water for 12 h; (c) 1 mM gibberellic acid (GA3) for 12 h; (d) 1 mM kinetin for 12 h; (e) 100 mM NaCl solution for 12 h; (f) PEG solution (40%) for 12 h or (g) for 24 h. The duration of priming was determined on the basis of our results on the kinetics of hydration and kinetics of germination (see the Results section). Seed priming was carried out in darkness at 20 °C on Whatman No. 1 filter paper in 9-cm diameter Petri dishes saturated with the treatment solutions. After priming, seeds were rinsed for 5 min with running tap water to remove priming solution from the seed coat and then dried under air flux at ambient temperature until they reached their initial moisture level (5.4% in Exagone, 5.8% in Toccata).
Seeds were then evaluated for germination in control and stress conditions (PEG Ψs = −0.9 MPa or NaCl 350 mM) as described previously. For each of the two cultivars, three replicates of 50 seeds per treatment were incubated on absorbent paper (filter paper circles, 8.5 cm diameter, Whatman Grade 1). The number of germinated seeds was counted every day for 7 days. Results were expressed in terms of G and T50.
Enzyme activities and total soluble sugar concentration
For protease extractions, 0.2 g of seeds were ground in liquid nitrogen in the presence of 1.2 mL homogenisation buffer (50 mM of Tris-HCl, 2 mM of ethylene diamine tetra-acetic acid (EDTA) and 5 mM of dithiothreitol (DTT)). The extract was centrifuged at 5000 g for 5 min at 2–4 °C and then at 25,000 g for 30 min at 2 °C. Supernatants were collected for further analysis. Proteases activity was measured with Protease Assay Kit (Calbiochem®) according to the manufacturer's instructions. A volume of 100 μL of the supernatant was added to 50 μL FTC-casein and 50 μL incubation buffer and incubated at 37 °C overnight. The reaction was stopped by adding 500 μL of 5% trichloroacetic acid (TCA) and samples were incubated at 37 °C for 10 min. The TCA precipitate was then pelleted by centrifuging at 12,000 g for 5 min. Finally, a volume of 600 μL of assay buffer was added to 400 μL of the supernatant and absorbance was read at 492 nm against the reagent blank.
For α-amylase extraction, 1 g of seeds were grounded and homogenised in 20 mL of 0.05 M succinate buffer (pH 5.2) containing 2 mM CaCl2. The extract was centrifuged at 10,000 g for 20 min at 3 °C and was filtered through two layers of Miracloth. The filtrate was immediately used as a crude enzyme preparation to determine α-amylase activity using a 0.2% (w/v) boiled starch solution and 0.5 mL succinate buffer. Samples were then incubated for 30 min at 30 °C, and 1 mL each of dinitrosalicylic reagent and distilled water were then added. The reaction mixture was heated for 5 min at 100 °C; after dilution by 5 mL of water, samples were read at 540 nm.
The β-amylase was extracted from 1 g of seeds ground in 10 mL of 50 mM Tris-HCl buffer at pH 7.0 containing 3 mM CaCl2, 4 mM NaCl, 0.1 mM phenylmethylsulphonyl fluoride (PMSF) and 0.1% Triton X-100. The extract was centrifuged at 9000 g for 1 h at 4 °C and the supernatant was subjected to precipitation with (NH4)2SO4 to reach 25% saturation at 4 °C, allowed to stand for 30 min at 0 °C and then centrifuged at 9000 g for 30 min. The supernatant was again treated with (NH4)2SO4 at 60% saturation: the solution was left at 0 °C for 30 min and then centrifuged at 9000 g for 30 min. The resulting precipitate was dissolved in 1 mL of extraction buffer (without Triton X-100) and dialysed overnight against the same buffer at 4 °C. The β-amylase was assayed according to Das and Sen-Mandi (Reference Das and Sen-Mandi1992): 0.2 mL of crude enzyme extract was incubated with 0.2 mL of starch substrate (1% soluble potato starch dissolved in 50 mM Na-citrate buffer (pH 3.6), 0.01% KH2PO4 and 1 mM EDTA) at 30 °C for 5 min. At the end of the incubation, 0.5 mL of dinitrosalicylic reagent and 1 mL of distilled water were added and the reaction mixture was heated for 5 min at 100 °C; after dilution by 5 mL of water, samples were read at 540 nm.
Both α- and β-amylase activities are expressed as units per mg protein, one unit being defined as the amount of enzyme hydrolysing 1 mg of starch for 1 min at 30 °C. Protein concentration in the extracted samples was determined according to Bradford (Reference Bradford1976).
Total soluble sugar concentration extracted from 1 g of ground seeds in 80% ethanol was measured by the Anthrone reagent method using glucose as the standard (Yemm and Willis, Reference Yemm and Willis1954) using a spectrophotometer (Beckman DU-640); the concentration of total soluble sugars in each sample was calculated according to a calibration curve achieved with a range of glucose concentrations from 0 to 500 mg L−1.
Statistical analysis
Percentage data were arcsin square root-transformed prior to analysis. Back-transformed means are given in tables and text wherever appropriate. Data were subjected to three- or two-way ANOVA. The means corresponding to the significant interaction of the highest order were reported in tables and compared by using the Least Significant Difference (LSD) at p = 0.05. The Multiple Comparison Procedure (MCP) was selected considering that the experimental design supports the adoption of a comparison-wise error rate controlling test (few comparisons of interest, mainly each treatment against control) (Onofri et al., Reference Onofri, Carbonell, Piepho, Mortimer and Cousens2010). Moreover, preliminary analysis showed that the adoption of other more conservative MCPs did not change our conclusions.
RESULTS
The presence of PEG or NaCl in the germination medium reduced the germination rate and final germination percentage in both Exagone and Toccata, but the effect was much greater in the latter (Table 1). For Exagone, NaCl up to 325 mM had no impact on germination and a little effect was recorded at 350 mM; only 400 mM increased T50 (5.2 days) and decreased G (75%). On the contrary, the effect of 350 mM was very marked in Toccata and seed germination was completely inhibited at 400 mM. Indeed, Toccata showed a marked increase in T50 (6.2 days) and a decrease in the final germination percentage (70%) already at 325 mM of NaCl (data not shown). Similarly, a matric potential of −0.9 MPa induced by PEG increased T50 (5.1 days) but did not reduce G (96%) in Exagone, while it strongly depressed germination in Toccata (T50 = 8.0 days, G = 26%).
Rate of germination (T50, i.e. number of days to reach 50% germination) and final germination percentage (G) of Exagone and Toccata sown on filter paper saturated with distilled water (H2O), 350- and 400-mM NaCl solution and PEG solution (Ψs = −0.9 and −1.0 MPa).
Values are means of three replicates. Arcsin square root-transformed data for G are reported in brackets.
LSD (p = 0.05; d.f. = 12) = NaCl: 4.35 for T50 and 10.1 for arcsin square root-transformed values of G.
PEG: 3.5 for T50 and 6.5 for arcsin square root-transformed values of G.
The presence of inhibitors in the germination substrate affected germination differently depending on the nature of the inhibitor, the cultivar and the solution used for saturating the filter paper (Table 2). Cx always inhibited germination, regardless of the cultivar or germination medium, and no seed germinated in its presence at −0.9 MPa PEG. Hx inhibited germination in both cultivars at −0.9 MPa PEG. CD decreased germination only at −0.9 MPa PEG, especially in Toccata, while the presence of Act-D completely inhibited germination only at −0.9 MPa PEG in both cultivars.
Rate of germination (T50, i.e. number of days to reach 50% germination) and final germination percentage (G) of Exagone and Toccata sown on filter paper saturated with distilled water (H2O), 350-mM NaCl solution and PEG (Ψs = −0.9 MPa) without any inhibitor (control) or in the presence of 100-μM hydroxyurea (Hx), actinomicin D (Act-D), cycloheximide (Cx) and cytochalasine (CD).
Values are means of three replicates. Arcsin square root-transformed data for G are reported in brackets. T50 for PEG with Act-D and Cx cannot be calculated since G = 0.
LSD (p = 0.05; d.f. = 60) = 0.69 for T50 and 6.1 for arcsin square root-transformed values of G.
Priming treatment also had different impacts on germination depending on the germination substrate (i.e. distilled water, NaCl or PEG solution), the priming treatment and the cultivar (Table 3). In control conditions, all primed seeds from any priming treatment germinated 1 day before non-primed ones irrespective of the cultivar or the priming treatment (Table 3). Priming treatments had only little or no effect on the germination of Exagone in the presence of NaCl. In Toccata, all priming treatments reduced germination except for GA3-priming in the presence of NaCl that caused an insignificant increase in G.
Rate of germination (T50, i.e. number of days to reach 50% germination) and final germination percentage (G) of Exagone and Toccata seeds non-primed or primed in (i) distilled water, (ii) NaCl 100 mM, (iii) gibberellic acid (GA3) 1 mM, (iv) kinetin 1 mM or (v) PEG (40%) solution for 12 h or 24 h. Seeds were then sown on filter paper saturated with distilled water (H2O), 350-mM NaCl or PEG solution (Ψs = −0.9 MPa).
Values are means of four replicates. Arcsin square root-transformed data for G are reported in brackets.
LSD (p = 0.05; d.f. = 126) = 0.92 for T50 and 7.6 for arcsin square root-transformed values of G.
Protein concentration (Figure 1a) and protease activity (Figure 1b) at 24 h from the start of imbibition did not differ significantly between cultivars and germination conditions. In both cultivars, a decrease in protein concentration was observed 24 h after the start of imbibition with respect to dry seeds and such a decrease was similar for H2O-, NaCl- or PEG-treated seeds.
(a) Protein concentration in Exagone and Toccata at dry seed state and after 24 h of imbibition, and (b) proteases activity after 24 h of imbibition in filter paper saturated with distilled water (H2O) or 350 mM NaCl solution (NaCl) or PEG solution (Ψs = −0.9 MPa). Values are means of five replicates. Vertical bars indicate ± SE of the mean.
The total soluble sugar concentration significantly increased in Exagone germinating in the presence of NaCl (Figure 2a). On the contrary, no significant increase was observed in Toccata in both stressing conditions. The α-amylase (Figure 2b) and β-amylase (Figure 2c) activities were generally decreased by salt stress and PEG only. The decrease of α-amylase was significant in Toccata for both salt stress and PEG, and in Exagone for PEG. Decrease of β-amylase activity was significant in both cultivars for both stresses. The α-amylase activity in dry GA3-primed and hydro-primed seeds was higher than in dry non-primed seeds for both cultivars (Figure 3) and it increased to maximum values in germinating seeds after 24 h of imbibition when germination occurred in stressing conditions, although values were similar for both cultivars.
(a) Total soluble sugars concentration, (b) α-amylase and (c) β-amylase activities in Exagone and Toccata seeds incubated for 24 h in filter paper saturated with H2O or 350 mM NaCl solution (NaCl) or PEG solution (Ψs = −0.9 MPa). Values are means of five replicates. Vertical bars indicate ± SE of the mean.
The α-amylase activity in Exagone and Toccata seeds non-primed and primed in (i) distilled water or (ii) water added with gibberellin (GA3): (a) dry seeds, and (b) seeds after 24 h of incubation on filter paper saturated with 350 mM NaCl solution or PEG solution (Ψs = −0.9 MPa). Values are means of four replicates. Vertical bars indicate ± SE of the mean.
DISCUSSION
The present work demonstrates that the germination of Exagone was more tolerant to salt and matric stresses than that of Toccata. In the presence of high salt concentration (325 mM NaCl corresponds to 50% of the salt concentration of sea water), Exagone performed even better than some halophyte species (Vicente et al., Reference Vicente, Boscaiu, Naranjo, Estrelles, Bellès and Soriano2004). In contrast, Toccata was sensitive to both types of constraints, thus confirming previous stress classifications (Al-Thabet et al., Reference Al-Thabet, Leilah and Al-Hawass2004; Andalibi et al., Reference Andalibi, Zangani and Nazari2005; Willenborg et al., Reference Willenborg, Gulden, Johnson and Shirtliffe2004). A water potential of −0.9 MPa (i.e. the value that allowed more than 90% germination for Exagone) in a structured loam soil would correspond to 3–5% water (by volume) lower than at −0.7 MPa (i.e. the value that allowed more than 90% germination for Toccata), equivalent to 3–5 mm of water (30–50 m3 ha−1 in the top 0.10 m soil layer). In Italy, with a sowing date in half September and an average evapotranspiration of around 0.5 mm day−1 (www.ucea.it [accessed 12 May 2010]; Allen et al., Reference Allen, Pereira, Raes and Smith1998), it should take around 1 week to evaporate 3–5 mm. This means that stress-tolerant cultivars like Exagone could be sown in late summer in soils with moderate water deficiency as can occur after an occasional rainfall in August–September and/or trusting in occasional/little rainfall after sowing as can be expected to occur in September.
The 350 mM NaCl concentration corresponded to a measured osmotic potential of −1.52 MPa, which is much lower than the −0.9 MPa PEG that inhibited Exagone germination. The higher inhibitory effect of PEG than NaCl on germination has also been reported by Huang and Redmann (Reference Huang and Redmann1995) for barley and Brassica species, Hampson and Simpson (Reference Hampson and Simpson1990) for soft wheat, Gulzar and Khan (Reference Gulzar and Khan2001) for Aeluropus lagopoides and Alam et al. (Reference Alam, Stuchbury and Naylor2002) for rice. Several possible circumstances may explain this evidence. Unlike PEG, NaCl may readily cross the cell membrane into the cytoplasm of the cell unless an active metabolic pump prevents accumulation of ions (Katembe et al., Reference Katembe, Ungar and Mitchell1998). Entering ions lower the seed osmotic potential, which facilitates hydration of the seed by allowing a higher seed matric potential than the osmotic potential of the solution surrounding the seed (Almansouri et al., Reference Almansouri, Kinet and Lutts2001). On the other hand, since PEG does not enter the seed during imbibition, its actual concentration in the solution increases (and the actual water potential decreases) with time (Hardegree and Emmerich, Reference Hardegree and Emmerich1994). In addition, PEG may harden seed coats causing mechanical resistance to tissue expansion and radicle protrusion.
The inhibitory effect of Cx on germination (higher T50, lower G) of both cultivars in all germinating environments (Table 2) indicates that de-novo synthesis of proteins is required for the germination of rapeseed. However, the lack of differences in protein concentration and protease activity (Figure 1) between treatments (substrates and cultivars) would suggest that protein metabolism is not directly implicated in rapeseed response to stress. Indeed, Prisco and Viera (Reference Prisco and Viera1976) assumed that the delay in the breakdown of protein in Vigna seeds germinating in saline substrate was due to the inhibition of translocation of hydrolysed products rather than to reduced protease activity. However, Kumar et al. (Reference Kumar, Sana and Hossain2004) reported that salinity stress reduced protease activity in Brassica seeds and similar evidence was reported by Ashraf et al. (Reference Ashraf, Afaf, Qureshi, Sarwar and Naqvi2002) for cotton varieties. Further research is needed in rapeseed to evaluate whether stress influenced inducible proteins for germinating seeds of tolerant and sensitive cultivars, as already found by Dell'Aquila and Spada (Reference Dell'Aquila and Spada1993) in Pisum sativum.
Act-D had no impact on seeds germinated in the presence of water, thus suggesting that gene transcription is not required in these conditions and that the Cx deleterious impact mainly concerns translation of long-lived mRNA already present in mature dry seeds. To the best of our knowledge, no literature is available on this aspect for rapeseed, but similar results have been observed in other species such as wheat (Jendrisak, Reference Jendrisak1980), lettuce (Schultz and Small, Reference Schultz and Small1991), sunflower (Lenormand et al., Reference Lenormand, Martini and Balangé1993), Arabidopsis thaliana (Rajjou et al., Reference Rajjou, Gallardo, Debeaujon, Vandekerckhove, Job and Job2004) and Poa pratensis (de Lespinay et al., Reference de Lespinay, Lequeux, Lambillotte and Lutts2010). The inhibitory effect of Hx on germination when seeds were incubated in −0.9 MPa PEG as compared with distilled water indicates that DNA replication is important for germination under matric stress. Since the inhibition of germination by Hx was much greater in Toccata than in Exagone, this would suggest that Exagone tolerance during germination reflects a lower amount of damaged DNA (Górnik et al., Reference Górnik, Castro, Liu, Bino and Groot1997).
Another explanation suggested by the reduced germination of Toccata at −0.9 MPa PEG in the presence of CD would be that radicle elongation in stress-resistant cultivar Exagone relies on cell elongation rather than cell division and thus does not require DNA replication for first germination steps. The inhibitory effect of Act-D for both cultivars at −0.9 MPa indicates that, in contrast to what occurs in the presence of water, the synthesis of mRNA is an essential requirement for germination under low water potential (Li et al., Reference Li, Feng, Han and Zhu2006; Rajjou et al., Reference Rajjou, Gallardo, Debeaujon, Vandekerckhove, Job and Job2004).
Increase in soluble sugar concentration found in Exagone seeds germinating in stressing conditions (Figure 2) as compared with both seeds germinating in distilled water and Toccata seeds in any substrate is probably related with salt and water stress tolerance, as already found in soybean (Meyer and Boyer, Reference Meyer and Boyer1981), wheat (Kameli and Lösel, Reference Kameli and Lösel1996) and chickpea (Gupta et al., Reference Gupta, Singh, Kaur and Singh1993). Actually, increase in sugar levels of stressed seed tissues might play an important role in osmotic regulation under water- and salt stress conditions as it does for mature plants (Jones and Turner, Reference Jones and Turner1980; Nemati et al., Reference Nemati, Moradi, Gholizadeh, Esmaeili and Bihamta2011). Increase in soluble sugar concentration in stressed tissues was puzzling since α- and β-amylase activities were reduced as a consequence of osmotic (NaCl) and matric (PEG) stresses (Figure 2b and c). Decrease in α-amylase activity at increasing salinity was also observed by Ashraf et al. (Reference Ashraf, Afaf, Qureshi, Sarwar and Naqvi2002) in seeds of several cotton varieties. It may be postulated that an important proportion of soluble sugars issued from amylase activities in tolerant cultivars could be used to lower osmotic potential and allow radicle protrusion through turgor and thus cell elongation maintenances. In a sensitive genotype, such as Toccata, sugars could have been used to produce energy as an unsuccessful attempt to overcome metabolic inhibition and diverted to activate biochemical processes such as DNA replication and/or repair. Nevertheless, difference in the sensitivity to inhibitors between two considered cultivars or between stress conditions could not be ruled out.
Faster germination for primed seeds germinating in distilled water (Table 3) was in line with the findings of other authors (Thornton and Powell, Reference Thornton and Powell1992; Tiryaki, Reference Tiryaki2006). Priming clearly hastened final germination and this could not be due to a higher initial water concentration, since the seeds were dried back to their original moisture concentration after priming. The general lack of effect of most priming techniques on germination under stress conditions and even the negative effect on G observed in Exagone germinating under low water potential were not expected and are in contrast with literature available for other rapeseed cultivars (Afzal et al., Reference Afzal, Aslam, Hameed and Ahmad2008; Zheng et al., Reference Zheng, Gao, Wilen and Gusta1998). This suggests that the ability of a given species to respond to priming treatment strongly varies among available cultivars.
CONCLUSIONS
The difference between the two rapeseed cultivars used in our experiment for response to stressing agents in the germination substrate was of the order of 0.2 MPa PEG for matric potential and 75 mMol for NaCl concentration. Such a difference could have an important agronomic impact on the establishment of rapeseed crops in dry and coastal environments such as Mediterranean regions. In the presence of matric (PEG) stress, Hx and CD inhibited germination only for the stress-sensitive cultivar. In the presence of osmotic (NaCl) stress, total soluble sugar concentration increased in seeds of the stress-tolerant cultivar. Based on these results, we speculate that stress tolerance to low water potential could result from a lower amount of damaged DNA in seeds, a lower requirement for DNA replication due to cell elongation rather than cell division involvement in radicle elongation and a higher soluble sugar concentration in stressed tissues leading to osmotic adjustment and maintenance of turgor. Priming techniques improved seed germination in non-stressing conditions, but had no effect or even negative effects on germination in stressing conditions.
Acknowledgements
We gratefully acknowledge Dr. Andrea Onofri for statistical support, and Mr. Silvano Locchi for technical assistance.
