Acta Biologica Hungarica 60 (4), pp. 433–439 (2009) DOI: 10.1556/ABiol.60.2009.4.9

INTERACTIVE EFFECTS OF SALINITY, NITRATE, LIGHT, AND SEED WEIGHT ON THE GERMINATION OF THE HALOPHYTE CRITHMUM MARITIMUM A. ATIA,*,** A. DEBEZ, M. RABHI, A. SMAOUI and C. ABDELLY Laboratoire d’Adaptation des Plantes aux Stress Abiotiques, Centre de Biotechnologies a` la Technopole de Borj Cedria, BP 901, Hammam-Lif, 2050, Tunisia (Received: February 13, 2008; accepted: November 26, 2008)

Interaction of salinity, nitrate, light, and seed weight on the germination of Crithmum maritimum was investigated. Seeds of three size categories were germinated at 0–200 mM NaCl with either 0, 5 or 20 mM KNO3. Experiments were done under darkness, white light, or red light. Regardless of seed weight, germination was maximal in distilled water. Under salinity, the smallest seeds showed the highest germination percentage. Salt impact was amplified by darkness, but was mitigated by nitrate supply, red light and their combination. At the same PPFD, germination of T2 seeds was higher, when exposed to red light than under white light, suggesting that germination was more influenced by the light type than by the PPFD. As a whole, not only salinity, nutrient availability, seed weight, and light, but also their interaction may control the germination of this halophyte. Keywords: Crithmum maritimum – germination – halophyte – light – nitrate – salinity

INTRODUCTION Despite halophytes are native plants of saline areas, they may be as salt sensitive as glycophytes at the early stages of their life cycle. Their germination is also affected by the combination of several factors including light [3, 16], nitrogen availability [11, 12], and seed size [6, 8]. Crithmum maritimum, or sea fennel (Apiaceae), is a fleshy aromatic, perennial littoral halophyte. It shows considerable economical and medicinal potentials [14], and its seeds contain appreciable amounts of oil, potentially edible, as the fatty acid composition is close to that of olive oil [15]. Data on the establishment requirements of C. maritimum in its natural habitats are scarce, we investi* Corresponding author; e-mail: [email protected] ** Current address: Institut für Botanik, Universität Hannover, Herrenhäuser Str. 2, D-30419, Hannover, Germany. 0236-5383/$ 20.00 © 2009 Akadémiai Kiadó, Budapest

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gated the effects of salinity, seed weight, light, nitrate, and their respective interaction on the germination of this oilseed halophyte.

MATERIALS AND METHODS Seeds collected from the rocky coasts of Tabarka (N-W of Tunisia) were separated in three classes (T1, T2, and T3) according to their individual weight: 1.5–2.5 mg, 2.6–3.5 mg, and 3.6-4.5 mg, respectively. Seeds were disinfected for 5 min in a 3.5% Ca(ClO)2 solution and sown in Petri dishes (25 seeds per each) containing a double layer of filter paper moistened with 2 ml of 0, 100, or 200 mM NaCl, with each solution containing either 0, 5, or 20 mM KNO3. Each of these 9 treatments was divided into three further lots (darkness, white light, or red light). Total darkness was ensured by covering the Petri dishes with four layers of black plastic film in a dark room. White light was produced by five fluorescent lamps (Type OSRAM 40 W, 25 µmol m–2 s–1, 400–700 nm), and red light was produced by the same lamps covered by a double layer of red Plexiglas (660 nm), so that the inactive (Pr) is converted to the active (Pfr) form of phytochrome [9]. In order to distinguish between the effect of the light wavelength and the light PPFD, T2 seeds were exposed for 2 h (at 0, 3, 7, 10, 14, 18, and 22 days) to a Pulse of red light or white light at the same PPFD. Two fluorescent lamps (Type OSRAM 15 W, 16 µmol m–2 s–1, 400–700 nm) filtered with a double layer of red Plexiglas or a triple layer of white Plexiglas, were used to produce, respectively, red and white light at a PPFD of 0.34 µmol m–2 s–1. All the experTable 1 Results of multi-way ANOVA of the effect of salinity, nitrate, light, seed weight, and their interactions on the final germination percentage of C. maritimum Parameter

F

P

Seed weight Nitrate Light Salt Nitrate * Seed weight Light * Seed weight Light * Nitrate Light * Salt Nitrate * Salt Seed weight*Salt Light * Nitrate * Seed weight Light * Nitrate * Salt Light * Seed weight * Salt Nitrate * Seed weight * Salt Light * Nitrate * Seed weight * Salt

52.736 28.241 117.911 334.399 1.198 0.144 1.919 5.428 3.320 12.793 1.146 2.043 2.641 0.285 0.753

0.000 0.000 0.000 0.000 0.295 0.99 0.066 0.000 0.023 0.000 0.333 0.115 0.014 0.998 0.848

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iments were carried out in a growth chamber at a temperature regime of 18 ± 5 °C and at an 8 h dark-16 h light photoperiod. Germination was recorded every alternate day. Seeds were considered to have germinated at the radicle emergence. A four-way ANOVA statistical analysis was carried out using SPSS 10.0 for Windows to determine the significance of individual and interactive effects of all factors (salinity, light, nitrate, seed size). Germination data (4 replicates per treatment) were arcsine transformed before analysis and a Tukey’s test (P < 0.05) to determine differences between means of the rate of germination.

RESULTS AND DISCUSSION The four-way ANOVA test indicated that the final germination percentage of C. maritimum was significantly affected by salinity, nitrate, light, and seed weight (Table 1). This was also true for the interactive effects of salt × nitrate (at P < 0.05), light × salt, salt × seed weight (at P < 0.0001) and light × seed weight × salt (at P < 0.05) (Table 1). Germination percentage of C. maritimum was maximal in distilled water but was significantly reduced at 100 mM NaCl and was virtually suppressed at 200 mM (Fig. 1A). Despite this response is common between halophytes and glycophytes, the salt tolerance threshold at the germination step varies greatly among halophytes. While the germination of Cakile maritima and Zygophyllum simplex was strongly impaired at salinities exceeding 100 mM NaCl [2, 13], it was maintained even at 500 mM NaCl in Aeluropus lagopoides [4]. Interestingly, Allenrolfea occidentalis germinated up to 800 mM NaCl [5]. The germination of C. maritimum was also seed size-dependent: while T1 seeds maintained a high germination capacity (>60%) at 100 mM NaCl, it was less than 40% in T2 and T3 seeds (Fig. 1A). This is consistent with previous reports on dimorphic seeds of Atriplex prostata and A. patula [8]. Small seeds may have germinated faster than the large seeds following a higher imbibition rate due to their small size. Seed size variation is regarded as a mechanism by which species extend the germination period over time, in order to avoid environmental constraints, and thereby increase the probability of the seedling establishment [4]. This could be particularly significant in environments subjected to regular perturbations of salt levels, such as rocky coasts. Germination was improved by nitrate under salt conditions, especially for T2 and T3 seeds (Fig. 1A, B, C), as documented in A. griffithii [11], Sporobolus arabicus [12], and Z. simplex [13]. In addition, the strong inhibition of germination under saline and darkness was partially overcome by the nitrate addition (Fig. 1G, H, I). Red light seemed more beneficial than white light, especially under salt stress, while darkness resulted in a strong reduction of the final germination percentage (Fig. 1A, D, G), confirming previous studies on halophytes [10, 16]. The likely lower gibberellic acid synthesis under darkness may partly explain such a finding [1]. Combining both nitrate and red light also enhanced the germination under salt conditions (Fig. 1A, E, F). The beneficial effect of red light was due to the wavelength Acta Biologica Hungarica 60, 2009

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Fig. 1. Interactive effects of NaCl, nitrate supply, and light conditions on the final germination percentage of the halophyte C. maritimum. Means within each histogram that have different letters are significantly different at (P < 0.05). T1: small seeds, T2: medium seeds, and T3: large seeds

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Fig. 2. Characterization of the wavelength and PPFD effects on the germination of C. maritimum. T2 seeds were either exposed to red light (R) or white light (W) for 2 h at (0, 3, 7, 10, 14, 18 and 22 days). (2A) Kinetics of germination, (2B) final germination percentage. Means that have different letters are significantly different at P < 0.05 (Tukey’s test)

rather than the PPFD, in fact when seeds were treated with a short pulse of red light or white light with the same PPFD, since red light exposure led to the highest germination under salt conditions (Fig. 2A, B). An integrative model was proposed to elucidate the effects of temperature, nitrate, and light on seed germination [7]. The three factors would act on a common site, the plasma membrane, where nitrate is fixed and increases the affinity of the protein receiver of the phytochrome, which in turn, attracts the active form of the phytochrome (Pfr). The formation of this complex (active receiver-form of the phytochrome) triggers signals of transduction leading to GA biosynthesis. As a whole, our results point out that C. maritimum germination is tightly governed by the seed size, salinity, nitrate and light availability, and their interaction. This may be important in the maintenance of the plant distribution in rocky coast ecosystems.

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