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Adaptive drought tolerance during germination of Salsola drummondii seeds from saline and non-saline habitats of

the Arid Arabian Deserts

Journal: Botany

Manuscript ID cjb-2018-0174.R1

Manuscript Type: Article

Date Submitted by the Author: 07-Nov-2018

Complete List of Authors: Elnaggar, Attiat; Universidad de Málaga, Departmento de Biología VegetalEl-Keblawy, Ali; University of Sharjah , Applied BiologyMosa, Kareem; University of SharjahNavarro, Teresa; Malaga University

Keyword: Drought tolerance, Germination requirement, maternal salinity, polyethylene glycol, Seed dormancy

Is the invited manuscript for consideration in a Special

Issue? :Not applicable (regular submission)

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1 ARESEARCH ARTICLE

2 Running title: Drought tolerance during germination in a habitat-indifferent halophyte

3 Adaptive drought tolerance during germination of Salsola drummondii seeds

4 from saline and non-saline habitats of the Arid Arabian Deserts

5

6 Attiat Elnaggar1,3,4, Ali El-Keblawy1, 2,*, Kareem A. Mosa1,5 and Teresa Navarro3

7 1Department of Applied Biology, Faculty of Science, University of Sharjah, PO Box 27272,

8 Sharjah, UAE

9 2Permanent address: Department of Biology, Faculty of Science, Al-Arish University, Egypt

10 3Departmento de Biología Vegetal, Universidad de Málaga, P. O. Box 59, 29080, Málaga, Spain

11 4Department of Botany and Microbiology, Faculty of Science, Alexandria University, Egypt.

12 5Department of Biotechnology, Faculty of Agriculture, Al-Azhar University, Cairo, Egypt

13 *Corresponding author Email: akeblawy@sharjah.ac.ae; phone +971505432065

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14 Abstract

15 The effects of temperature, light, salinity, and drought on germination of halophytes have been

16 extensively studied. However, few studies have focused on the germination of plants that grow

17 well in both saline and non-saline habitats (i.e., habitat-indifferent halophytes). Here, we assess

18 the impacts of population origin, temperature, and light on drought tolerance, as simulated by

19 polyethylene glycol (PEG), during germination of Salsola drummondii, a habitat-indifferent

20 halophyte from the arid Arabian deserts. Seeds were collected from both saline and non-saline

21 habitats and germinated at six PEG levels at three temperatures and two light regimes. An

22 increase in PEG concentration resulted in a significant reduction in seed germination, especially

23 at higher temperatures. Seeds from the non-saline habitat attained significantly greater

24 germination efficiency at PEG levels up to -1.2 MPa, but there was no difference in germination

25 of seeds between the two habitats at -1.5 MPa PEG concentrations. Saline habitat seeds

26 germinated significantly faster at higher PEG levels. Germination was significantly higher in

27 dark than in light at -1.5 MPa at lower temperatures, but the opposite was true at higher

28 temperatures. Seeds from saline habitats had higher dormancy and faster germination at higher

29 concentrations of PEG due to adaptation to low osmotic potentials.

30 Keywords: Drought tolerance; Germination requirement; maternal salinity; polyethylene glycol;

31 seed dormancy

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32 Introduction

33 Water will become a scarce natural resource with the increasing aridity and growing world

34 population, and this scarcity is expected to be more severe in arid and hyper-arid regions (Evans

35 2009). Several scenarios of climate-change predict an increase in water deficit and aridity in

36 many regions of the world, which necessitates research on plant tolerance to drought stress (Petit

37 et al. 1999). Drought leads to reduction in soil water content, and consequently, is one of the

38 most pernicious environmental stress factors facing plants (Cosgrove and Rijsberman 2014).

39 Plants respond to drought stress through different mechanisms that involve molecular,

40 biochemical, physiological, and morphological changes (Levitt 1972; Turner 1986; Mosa et al.

41 2018). Of the many possible tolerance mechanisms, plants commonly contend with drought

42 through osmotic adjustment and by balancing the ratio of different osmolytes (Flowers and Yeo

43 1986; Nounjan et al. 2018).

44 Germination and recruitment of desert halophytes are affected by environmental factors,

45 such as soil salinity, availability of water, temperature, and light intensity (Baskin and Baskin

46 2014). Such effects of environmental factors are especially obvious in the salt marshes of arid

47 deserts, where evaporation is very high (Khan and Weber 2000; El-Keblawy 2004; El-Keblawy

48 and Bhatt 2015; El-Keblawy et al. 2015). The scarcity of rainfall, coupled with the tendency of

49 soil salinity to increase, makes drought a serious problem facing halophytes of the Arabian arid

50 deserts, where annual average precipitation is very low (e.g., around 100 mm in the United Arab

51 Emirates, UAE, Böer 1997). Under such conditions, seeds of halophytes postpone their

52 germination until arrival of suitable conditions for seedling survival, which usually happens

53 when effective rainfalls increase soil water potential and other conditions, including especially

54 temperature and light, become suitable as well (Khan and Weber 2000; El-Keblawy 2004, 2014).

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55 Several studies have assessed the impacts of these factors - individually or in combination - on

56 salinity tolerance by plants during germination, but few have evaluated the effect of these factors

57 on drought tolerance of halophytes.

58 Environmental maternal effect, which is determined by conditions experienced by

59 maternal plants during seed maturation, often plays a substantial role in controlling the

60 germinability of ripening seeds (Roach and Wulff 1987). It has been reported that maternal

61 habitat and time of seed development affect seed dormancy and germination requirements (Siles

62 et al. 2017; El-Keblawy et al. 2017a, b, 2018; Al-Shamsi et al. 2018). Adaptive maternal effect

63 enhances the progeny’s fitness in an environment similar to that experienced by the maternal

64 plants (Rossiter 1996; Soliman et al. 2018). Several studies have assessed the impact of multiple

65 environmental factors (e.g., temperature, rainfall, light quality, and day length) during seed

66 development and maturation during seed germination (see Roach and Wulff 1987; Fenner 1991;

67 Wulff 1995; Gutterman 2000). However, few studies have assessed the effect of maternal

68 salinity on dormancy and germination requirements of habitat-indifferent halophytes (e.g., El-

69 Keblawy et al. 2016, 2017a, 2018). In Anabasis setifera, seeds from the non-saline habitat have

70 been reported to have significantly higher germination levels than those from the saline habitat at

71 all salinity levels (El-Keblawy et al. 2016). In Suaeda aegyptiaca and S. vermiculata, seeds from

72 the non-saline habitat attained significantly higher germination as compared to those from saline

73 habitat, in lower and moderate salinities. At higher salinities, however, the germination of seeds

74 from the saline habitat was either similar or higher than that for seeds from the non-saline habitat

75 (El-Keblawy et al. 2017a, 2018).Taking into consideration that water deficit (drought) is usually

76 associated with salt stress, it is important to assess the effect of maternal salinity on drought

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77 tolerance and germination requirements during seed germination of habitat-indifferent

78 halophytes (Al-Shamsi et al. 2018).

79 Salsola drummondii Ulbr. (family Amaranthaceae) is a perennial, leaf succulent, habitat-

80 indifferent xerohalophyte. In the United Arab Emirates (UAE), this evergreen species grows

81 equally well in both saline and non-saline soils. The plants associated with S. drummondii in the

82 saline soils are true halophytes (i.e., growing only in saline habitats) and include species such as

83 Aeluropus lagopoides, Halopeplis perfoliata, and Halocnemum strobilaceum. However, species

84 associated with S. drummondii in non-saline habitats are glycophytes, such as Launaea capitata,

85 Cornulaca monacantha, Pennisetum divisum, and Indigofera oblongifolia. Other habitat-

86 indifferent halophytes, such as Suaeda vermiculata and Zygophyllum qatarense, are also

87 recorded as being associated with S. drummondii in the two habitat types (Jongbloed 2003).

88 There are several economic uses of this species. For example, leaves can be burnt to produce

89 soda ash. In addition, different parts of this plant have medicinal uses (Gilani et al. 2010). The

90 leaves can also be used for feeding animals (Qureshi et al. 1993). Furthermore, S. drummondii is

91 an important plant for the restoration of salt-affected or degraded habitats (Dagar and Minhas

92 2016).

93 Both drought and salinity stresses reduce soil water potential. Polyethylene glycol (PEG) can

94 be used to create solutions with various negative water potentials (Money 1989). Drought usually

95 affects plants through affecting soil water potential, i.e., osmotic effect. However, the effect of

96 salinity could be mediated through specific ion toxicity and/or reduced soil osmotic potential

97 (Munns 2002; Kranner and Seal 2013; Maucieri et al. 2018). Polyethylene glycol has been used

98 to simulate drought or water-stress during germination and plant growth (Kołodziejek and

99 Patykowski 2015). Germination inhibition in PEG-treated seeds is attributed mainly to osmotic,

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100 rather than ion-specific toxicity effects, which is usually attributed to salinity. Several

101 researchers used this solute in germination studies to detect whether the effect of salinity on

102 certain species is osmotic and/or ion-specific (Tobe et al. 2000; Sosa et al. 2005; Hameed et al.

103 2013). The marked differences in germination response observed at the same osmotic potentials

104 with NaCl and PEG indicate ionic-specific effects (Sidari et al. 2008). In a Pakistani population,

105 Rasheed et al. (2015) studied germination of S. drummondii and reported that its seeds can

106 germinate in up to 1000 mM NaCl (i.e., almost twice as high as seawater salinity). Tolerance to

107 salinity level differs based on both temperature and light conditions during germination (Rasheed

108 et al. 2015). The survival of S. drummondii in both saline and non-saline habitats makes it a good

109 model for assessing the effect of maternal habitat on seeds’ response to drought and dormancy

110 during germination. As development and maturation of S. drummondii seeds in saline habitats

111 are subjected to both salt and, possibly, water deficit stress of the arid environment, we

112 hypothesize that seeds collected from saline habitats may have a greater drought tolerance as

113 compared to seeds from non-saline habitats, which could be subjected only to water deficit

114 stress. Therefore, the goal of this study was to assess the response of drought tolerance, as

115 simulated by PEG, during the germination stage of S. drummondii seeds collected from saline

116 and non-saline habitats in the presence of different light and temperature regimes. Higher

117 drought tolerance is expected in seeds from saline habitat as this characteristic helps them

118 survive the higher salinity levels of their natural habitats. In addition, we have assessed the

119 significance of the interaction between the main factors (i.e., maternal habitat, drought, light, and

120 temperature) to determine the dependence of drought tolerance on light and temperature during

121 germination of seeds from the two habitat types. This, in turn, will help determine the proper

122 time of germination under natural conditions of seeds from the two habitats. Moreover, it is

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123 important to define a seed population that can tolerate higher drought levels during germination

124 for a species that has the potential to be used in restoration of degraded deserts. The seeds of the

125 habitat type that can tolerate higher drought can be used for restoration purposes even during

126 years that receive little rainfall.

127

128 Materials and methods

129 Study site and seed collection

130 Mature seeds of Salsola drummondii were collected from two sites around Kalba city, the eastern

131 coast of the UAE, during December 2015. One site was saline with compacted surface soils

132 (24°99′68.68″N and 56° 34′91.90″E) and the other was a non-saline sand plain (25° 02′94.17″N

133 and 56° 36′17.56″E). The saline and non-saline soil types could be classified according to the

134 USDA soil taxonomy as Haplosalids and Typic Torripsamments, respectively (Shahid et al.

135 2014). Seeds of each habitat were randomly collected from 50–60 plants. Collected seeds were

136 cleaned and stored in brown paper bags at -18 °C until the experiment was initiated in the first

137 week of January 2016. At that time of the year, effective rainfalls usually occur and therefore

138 germination takes place.

139 Five soil samples were collected from around S. drummondii at each habitat. Soil samples

140 were air dried and sieved. Soil salinity, electrical conductivity (EC), and pH were measured from

141 a 1:5 soil: water suspension (Dahnke and Whitney 1988). After 24h of shaking, the suspension

142 was left undisturbed for 1h, then pH and electrical conductivity (EC) were measured by Thermo

143 Scientific Orion Star A211 pH Benchtop Meter. Salinity was measured using a HQ40d salinity

144 meter (HACH, Loveland, Colorado, USA).

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145 Effects of maternal salinity on drought tolerance at different light intensities and temperatures

146 Polyethylene glycol is an inert polymer. It is nonionic, has high molecular weight, and can be

147 dissolved in water. It is the most preferred osmotic substance that can create solutions with

148 various negative water potentials. We used PEG to assess drought tolerance during germination

149 in order to determine the minimum water potential threshold for germination (Bradford 2002).

150 Solutions with higher levels of PEG have higher negative water potentials (Money 1989; Munns

151 2002). Several studies have used PEG to simulate drought during germination in several species

152 (Okçu et al. 2005; Sidari et al. 2008; Muscolo et al. 2014; Cochrane et al. 2015; Kołodziejek and

153 Patykowski 2015; Cavallaro et al. 2016). As PEG is a non-penetrating polymer, it affects seed

154 germination through its osmotic effect (Munns 2002).

155 To assess drought tolerance during germination of seeds from both the saline and non-

156 saline habitats and to estimate the dependence of the tolerance on incubation temperature and

157 light regimes, seeds from the two habitat types were germinated in six PEG 6000 (Sigma-

158 Aldrich) levels (0, -0.4, -0.7, -1.0, -1.2 and -1.5 MPa) and incubated in three CONVIRON plant

159 growth chambers (model E-15) adjusted at three temperatures, each with two light regimes. The

160 three temperatures were 15/25, 20/30 and 25/35°C with 12-h dark/12-h light cycles, where high

161 temperatures coincided with 12 hrs of white light. The two light regimes were light (12-hr

162 light/12-hr dark) and complete darkness (hereafter referred as light and dark regimes,

163 respectively). The PEG levels used here were selected after a preliminary test to assess the

164 drought tolerance of S. drummondii during germination. The osmotic potentials of the prepared

165 PEG solutions were verified using a Wescor Vapro 5520 (Wescor Inc., UT, USA) capable of

166 measuring osmotic potentials. The lighting in the chamber was white light (1400 µmol m–2 s–1 of

167 photosynthetically active radiation) provided by five (400 W) metal halide and five (400 W) high

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168 pressure sodium lamps. Germination was conducted in 9-cm Petri dishes on two layers of

169 Whatman No. 1 filter paper, moistened with 10 ml of the test solutions. As a precaution to

170 minimize evaporation, the plates were wrapped with parafilm. To achieve the dark treatment, the

171 dishes were wrapped in aluminum foil. Four replicate dishes, each with 25 seeds, were used for

172 each treatment. Visible radicle protrusion was used as an indication for germination. Under the

173 light regime, germinated seeds were counted every other day for 20 days following seed

174 imbibition. Seed viability in each habitat type was assessed for three batches, each consisting of

175 100 seeds, using 1% (w/v) 2,3,5- triphenyl-tetrazolium chloride solution (Bradbeer 1998).

176 Germination recovery

177 At the end of germination experiment (i.e., after 20 days), non-germinated seeds in the different

178 PEG solutions at different light and temperature regimes, were washed and placed in distilled

179 water to determine if they would germinate. This recovery experiment was conducted at the

180 same temperature regimes mentioned above and under the same light conditions. Germinated

181 seeds were counted every other day for 10 days.

182 Calculations and data analyses

183 The rate of germination was estimated by using a modified Timson index of germination

184 velocity: ΣG/t, where G is the percentage of seed germination at 2-day intervals, and t is the total

185 germination period (Khan et al. 2000). The maximum possible value in our data, using this

186 germination rate index, was 50. This value means that all germination occurred in the first count

187 (i.e., after two days).

188 The germination recovery percentage was calculated using the following formula (Khan et al.

189 2000): Recovery percentage = (a-b)/(c-b)*100, where a is the total number of seeds germinated

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190 after being transferred to distilled water, b is the total number of seeds germinated in PEG

191 solution, and c is the total number of seeds.

192 Four-way ANOVAs were used to evaluate the significance of the four factors (i.e., maternal

193 habitat, drought, temperature, and light) and their effects on final germination, germination

194 recovery, and total germination (germination in saline solution plus recovery germination).

195 Three-way ANOVA was used to assess the impacts of maternal habitat, drought, and

196 temperature and their interactions on germination rate index (GRI). Pearson correlation

197 coefficients were calculated to assess the relationship between PEG concentrations and total

198 germination. One way ANOVAs were performed to assess the significant differences between

199 the saline and non-saline habitats in EC, salinity and pH. Tukey’s test (Honestly significant

200 differences, HSD) was used to estimate the least significant differences between the means at P =

201 0.05. Germination percentages were arcsine-transformed to meet ANOVA assumptions. This

202 transformation improved the normality of the data distribution.

203 Results

204 Soil and seed properties of the two habitat types

205 Soils of the saline habitat attained significantly greater electric conductivity (EC) (21.3 mS/cm),

206 in comparison to the non-saline habitat (1.4 mS/cm, F = 270, P < 0.001). In addition, the pH was

207 significantly greater in soils of the saline habitat (pH = 9.0), compared to those of the non-saline

208 habitat (pH = 8.0, F = 83.0, P<0.001). Furthermore, salinity attained significantly greater values

209 in the saline (5.5 g/l) than in the non-saline habitats (0.38 g/l, F = 12.2, P<0.001). There was no

210 significant difference between the average seed mass of the saline (0.680 mg) and non-saline

211 (0.745 mg) habitats (F = 2.2, P>0.05). In addition, seed viability did not differ significantly

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212 between the seed lots of the two habitats (88.7% and 92.6%, for saline and non-saline habitats,

213 respectively, F = 3.2, P>0.05).

214 Final germination in PEG solutions

215 There were significant effects for the main factors and many of their interactions on the final

216 germination of S. drummondii (P<0.05, Table 1). The significant interaction between maternal

217 salinity and PEG concentration indicates that tolerance to drought as simulated by PEG

218 depended on seed source. No germination occurred at -1.8 MPa PEG for seeds from the two

219 habitat types. Seeds of the non-saline habitat attained significantly greater germination levels, as

220 compared to those of the saline habitat, in PEG concentrations up to -1.0 MPa (Fig. 1). The

221 difference diminished in -1.2 MPa PEG and completely disappeared in -1.5 MPa. This result

222 implies that seeds of the non-saline habitat had greater germination in higher osmotic potential,

223 compared to those of the saline habitat. At lower osmotic potentials (-1.5 MPa), however, there

224 was no significant difference in final germination between seeds of the two maternal habitats

225 (Fig. 1a).

226 The interactions between PEG treatment and both temperature and light were significant

227 (P<0.01, Table 1), indicating that the tolerance to PEG osmotic potential depended on these

228 conditions during seed sowing. For example, whereas no significant difference was observed in

229 final germination at lower (15/25 °C) and higher temperatures (25/35 °C) in distilled water and -

230 0.4 MPa PEG, germination was significantly greater at lower than at higher temperatures at PEG

231 levels ≥ -0.7 MPa (Fig. 1b). However, the effect of light on drought tolerance was not clear;

232 whereas germination was significantly greater in light than in dark in -0.7 and -1.2 MPa, there

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233 was no significant difference between germination in light and dark at the other PEG

234 concentrations (Fig. 1c).

235 Germination rate index in the PEG solutions

236 The effects of maternal salinity, PEG, and temperature on GRI were significant (P<0.001, Table

237 2). GRI decreased with increasing PEG concentrations; GRI was 49.6 in the control but reduced

238 to 32.1 at -1.5 MPa. There were significant effects of the interaction between PEG and both

239 maternal salinity and temperature of incubation on the GRI (P<0.001, Table 2). No significant

240 difference in GRI was observed between seeds of the two habitats at lower concentrations of

241 PEG (0, -0.4 and -0.7 MPa). However, seeds of the saline habitat attained significantly higher

242 GRI (i.e., germinated faster) in the higher concentrations of PEG (Fig. 2a). In addition, GRI did

243 not differ between the different temperatures at higher osmotic potentials (PEG levels ≤-1.0

244 MPa). However, at lower osmotic potential (PEG levels= -1.2 and -1.5 MPa), germination was

245 significantly faster at higher than at lower temperatures (Fig. 2b).

246 Germination recovery

247 There were significant effects of maternal salinity, drought, and temperature, but not light, on

248 germination recovery (P<0.001, Table 1). The significant effect of the interaction between

249 maternal salinity and PEG indicates that recovery of seeds from different PEG concentrations

250 depended on the seed source (i.e., maternal habitat). Germination recovery occurred mainly in

251 seeds that failed to germinate in -1.2 and -1.5 MPa and was significantly greater for seeds of

252 plants in non-saline habitats than for those of plants in saline habitats (Fig. 3a). In addition, the

253 response of germination recovery at different concentrations of PEG depended on temperature of

254 incubation; the interaction between PEG concentration and temperature was significant (P<0.01,

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255 Table 1). There was no significant effect for temperature on germination recovery in all osmotic

256 potential, expect in the lowest osmotic potential (-1.5 MPa), in which germination recovery was

257 significantly greater at lower than at higher temperatures (Fig. 3b). The interaction between PEG

258 and light was non-significant. However, recovery was greater for seeds incubated in light, as

259 compared to those in dark, in osmotic potential -1.2 and -1.5 MPa. (Fig. 3c).

260 Total germination

261 All the four main factors showed significant effects on the total germination (i.e., germination in

262 PEG solution plus germination recovery) (P<0.05). However, only three interactions between the

263 main factors were significant, compared to six significant interactions for germination in PEG

264 (Table 1). In addition, Pearson correlation coefficients assessing the relationship between total

265 germination and PEG concentrations indicated significant negative relationships at all

266 temperatures for seeds of the non-saline habitat (r = - 0.77, P<0.001 at 15/25 °C; r = - 0.59,

267 P<0.001 at 20/30 °C; r = - 0.65, P<0.001 at 25/35 °C). For seeds of the saline habitat, however,

268 the negative relationship was significant at 15/25 °C (r = - 0.50, P<0.001) and 20/30 °C (r = -

269 0.49, P<0.001), but not at 25/35 °C (r = - 0.04, P>0.05).This result indicates that the germination

270 of S. drummondii seeds of the saline habitats was less affected by the increase in PEG

271 concentration at higher temperatures, but seeds of the non-saline habitats were negatively

272 affected by the increase in the PEG concentrations at all temperatures.

273 Discussion

274 The results of our study showed significantly greater germination of seeds from the non-saline

275 habitat as compared with those from saline habitat in all PEG concentrations up to -1.2 MPa. At -

276 1.5 MPa; however, there were non-significant differences in the final germination of the two

277 seed lots. The results also showed insignificant differences in the viability of non-saline and

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278 saline habitat seeds. This indicates that seeds from the saline habitat are more dormant, as

279 compared to those from the non-saline habitats when germinated in lower osmotic potential

280 solutions of PEG. Maruyama et al. (2016) explained the higher dormancy observed in Impatiens

281 capensis seeds produced under maternal drought stress to be a mechanism for desiccation

282 avoidance/drought tolerance in heterogeneously dry sites. The greater dormancy of the saline

283 habitat seeds of S. drummondii helps them postpone their germination until the onset of

284 favorable conditions for seedling establishment. Such conditions usually occur when effective

285 rainfall happens at lower temperatures during winter (e.g., during November – February, El-

286 Keblawy 2004, 2017).

287 The combined effect of soil temperature and water content on seed germination is an

288 environmental signal that could determine the germination time (Fyfield and Gregory 1989). Our

289 results showed non-significant differences between the germination of seeds at the three

290 temperatures, when S. drummondii seeds were incubated at low levels of drought (0 and -0.4

291 MPa). At higher drought levels, however, germination was significantly reduced at high

292 temperatures (25/35 °C) as compared to lower temperatures (15/25 °C). This could be an

293 ecological adaptation to reduce germination at the end of the growing season, when conditions

294 are not favorable for seedling establishment (Hameed et al. 2013; El-Keblawy et al. 2015;

295 Rasheed et al. 2015). In general, exposing seeds to two stress factors (drought and high

296 temperatures) would affect the integrity of cell membranes (Raison 1986). Electrolyte leakage

297 resulting from loss of membrane integrity at higher temperatures reduced germination in several

298 species, such as Brassica spp. (Thornton et al. 1990) and Brassica olearcea (Jett et al. 1996).

299 Germination of S. drummondii was significantly greater under dark conditions than under

300 light at the highest concentration of PEG (-1.5 MPa), when seeds were incubated at lower

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301 temperatures (15/25 °C), but the reverse was true at higher temperatures (25/35 °C). This result

302 might be ecologically important for the survival of S. drummondii in arid desert conditions.

303 During dry years, when soil water potential is low, seeds germinate only when rainfalls occur at

304 cooler temperatures and seeds are allowed to germinate under conditions of darkness, e.g., while

305 being covered with litter or by remaining in a soil crack. Such conditions enhance seedling

306 establishment in arid deserts, where soils have lower rates of water retention (El-Keblawy 2017).

307 Both drought and salinity lower the plant water potential by reducing the free energy of

308 water available for the plant to be below that of pure free water. To avoid desiccation, the water

309 potential of the symplast must be adjusted (Flowers and Yeo 1986). Osmotic adjustment could

310 be through ion contents or production of organic osmolytes (Ghoulam et al. 2002). In a study

311 assessing salinity tolerance in S. drummondii, Rasheed et al. (2015) reported that some seeds

312 were able to germinate in 1000 mM NaCl (around -5.0 MPa). In the present study, however,

313 seeds of S. drummondii germinated only to less than 20% in -1.5 MPa PEG solutions. The

314 greater tolerance to lower osmotic potential resulted from NaCl than that from PEG has been

315 reported in several other species. In Ceratonia silique, for example, germination was

316 significantly reduced by a water stress simulated by a moderate level of PEG (−0.5 MPa), but it

317 occurred in higher levels of NaCl (−1.0 MPa, Cavallaro et al. 2016). Similarly, the effects of

318 NaCl on both germination and seedling growth of three pea cultivars was significantly less,

319 compared to the effect of PEG (Okçu et al. 2005). Furthermore, germination of Henophyton

320 deserti seeds was less affected by NaCl, as compared with PEG (Gorai et al. 2014).

321 The greater tolerance of S. drummondii to NaCl, compared to drought simulated by PEG

322 indicates that osmotic, rather than toxicity effect would be responsible for the failure of

323 germination of this species in saline solutions. The result also indicates that PEG might cause

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324 irreversible damage to the cells (Yu and Rengel 1999). The rehydration of desiccated seeds after

325 being treated with PEG could be associated with damage of the plasma lemma, and consequently

326 leakage of cell solutes (Hendricks and Taylorson 1976; Yu and Rengel 1999). Changes in

327 organelle morphology and function, and disruption of organelle membranes due to desiccation

328 associated with water stress have been also reported in other species (see Dhindsa and Bewley

329 1977). The ability of S. drummondii to tolerate and recover from -5.0 MPa NaCl (Rasheed et al.

330 2015), but only in -1.5 MPa PEG (our study) indicates that this species is more adapted to saline

331 than drought conditions. In fact, this species produce flowers and fruits before the onset of the

332 rainy season (October – November), despite the fact that it has a very shallow root system to tap

333 groundwater, indicating that it depends on atmospheric moisture as a non-conventional source

334 for water, in absence of soil water during the dry summer (Dirks et al. 2016).The ability of the

335 plants of S. drummondii to flower and fruit in the absence of conventional soil water indicates

336 that they are relying on moisture absorption from air rather than soil. Consequently, as seeds

337 have a limited ability to absorb atmospheric moisture, they cannot tolerate soil moisture

338 deficiency as adult plants do.

339 Climatic factors, in particular temperature and water availability, have a considerable

340 influence on plant recruitment and survival (Gurvich et al. 2017). These factors are critical

341 drivers for seed dormancy and germination. Consequently, plant recruitment and population

342 dynamics will certainly be affected by projected climate change (Walck et al. 2011). Evans

343 (2009) used 18 global climate models and predicted an overall temperature increase of ∼1.4 K

344 by mid-century, increasing to almost 4 K by late-century for the Middle East. In addition, he also

345 predicted increases in the length of the dry season and changes in the timing of the maximum

346 precipitation that will impact the growing season (Evan 2009). Our results showed that seeds of

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347 S. drummondii from both saline and non-saline habitats germinated to more than 40% under high

348 levels of drought (-1.2 MPa). In addition, more than 25% of the seeds germinated at high

349 temperatures (i.e. above the average of the growing season, Böer B 1997) (Fig. 1a,b). The broad

350 windows of germination under different temperature and light regimes and the ability of seeds to

351 germinate under relatively lower osmotic potentials indicate that S. drummondii is less

352 threatened by the projected climate change in the Middle East.

353 Plants are sedentary organisms that have little choice for the suitable environment where

354 they can grow and reproduce. Environmental stresses that are experienced by paternal plants can

355 induce phenotypic changes that span multiple generations (Münzbergová and Hadincová 2017).

356 Transgenerational plasticity provides phenotypic variation that contributes to adaptation to

357 environmental stresses (Vu et al. 2015). Transgenerational phenotypic plasticity in progeny traits

358 can occur through maternal and/or epigenetic effects (Soliman et al. 2018). Maternal effects in

359 plants include the maternal genetic effects caused by maternal inheritance of plastids in addition

360 to non-inheritance effect of endosperm, seed coat, resource provisioning of nutrient resources,

361 hormones, proteins and transcripts (Vu et al. 2015; Verslues 2016). Whereas environmental

362 maternal effects are usually diminished in the first generation, epigenetic effects transmit

363 heritable plastic responses to environmental cues (Uller et al. 2008). In our study, it is not clear

364 whether the differences in seed dormancy, germination responses and drought tolerance between

365 seeds from saline and those from non-saline habitats are due to maternal and/or epigenetic

366 effects. Therefore, further studies are needed to separate epigenetic effect from maternal salinity

367 effect in habitat-indifferent halophytes, such as S. drummondii. For example, reciprocal

368 transplant experiments between the two populations should be conducted and seed germination

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369 response and drought tolerance from these plants should be compared. Another approach could

370 be through growing micropropagated plants from the two populations in both habitat types.

371 Conclusion

372 Seeds of the non-saline habitats had significantly lower dormancy and higher germination in

373 PEG concentrations up to -1.2 MPa, as compared with seeds of the saline habitat. The relatively

374 higher dormancy of the saline habitat seeds indicates that they prefer to postpone their

375 germination until the arrival of proper conditions for seedling establishment, which is usually

376 after winter rainfalls in the arid Arabian deserts. The faster germination of saline habitat seeds in

377 lower osmotic potential solutions indicates that they establish themselves shortly after rainfalls,

378 especially in years that receive less than average rainfalls.

379 Acknowledgements

380 This work was partially supported through a grant from the University of Sharjah Research

381 Office that supported to the Environmental and Chemical Biology Research Group (Grant #

382 150404).The authors would like to thank Mr. Mohammed Hassan, Sharjah Research Academy

383 for his help in seeds collection.

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576

577 Table 1. Results of four-way ANOVA showing the effects of maternal salinity and environmental factors

578 during incubation (drought, as stimulated by PEG, temperature and light) and their interactions on final

579 germination in PEG solution, germination recovery after seeds transferred from PEG to distilled water

580 and total germination (i.e., germination in PEG solutions plus germination recovery) of Salsola

581 drummondii seeds

Final germination Recovery germination

Total germination

Source of variation df Mean Squares

F-Ratio Mean Squares

F-Ratio Mean Squares

F-Ratio

Maternal salinity (MS) 1 3.016 411.6*** 0.030 58.60*** 3.757 434.40***PEG 5 1.801 245.8*** 0.046 90.27*** 1.276 147.56***Temperature (T) 2 0.250 34.2*** 0.009 18.19*** 0.376 43.44***Light (L) 1 0.140 19.2*** 0.001 1.850 0.113 13.06***MS * PEG 5 0.158 21.5*** 0.016 31.98*** 0.067 7.71***MS * T 2 0.003 0.458 0.001 1.874 0.004 0.486MS * L 1 0.003 0.403 0.000 0.031 0.004 0.427PEG * T 10 0.024 3.22** 0.006 12.17*** 0.035 4.00***PEG * L 5 0.027 3.68** 0.000 0.948 0.024 2.72*T * L 2 0.068 9.29*** 0.001 0.996 0.077 8.90***MS * PEG * T 10 0.009 1.246 0.001 1.595 0.010 1.178MS * PEG * L 5 0.021 2.92* 0.001 1.597 0.020 2.136MS * T * L 2 0.002 0.322 0.002 3.37* 0.001 0.068PEG * T * L 10 0.021 2.9** 0.000 0.677 0.017 2.007MS * PEG * T * L 10 0.008 1.088 0.001 1.65 0.010 1.196Error 216 0.007 0.001 0.009

582

583 Table 2. Results of three-way ANOVA showing the effects of maternal salinity, and PEG concentration

584 and temperature and their interactions on germination rate index of Salsola drummondii seeds

Source of variation df Mean Squares F-Ratio PMaternal salinity (MS) 1 0.247 46.971 <0.001PEG 5 0.471 89.674 <0.001Temperature (T) 2 0.051 9.723 <0.001MS * PEG 5 0.055 10.404 <0.001MS * T 2 0.004 0.793 nsPEG * T 10 0.010 1.848 nsMS * PEG * T 10 0.005 0.920 nsError 108 0.005

585

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586 Figure captions

587 Fig. 1. Interactive effects of drought, as simulated using PEG, with (a) maternal salinity, (b)

588 temperature of incubation and (c) light of incubation on final germination percentage (mean ±

589 S.E.) of Salsola drummondii seeds.

590

591 Fig. 2. Interactive effects of drought, as simulated using PEG, with (a) maternal salinity and (b)

592 temperature of incubation on germination rate index (mean ± S.E.) of Salsola drummondii seeds.

593

594 Fig. 3. Interactive effects of drought, as simulated using PEG, with (a) maternal salinity, (b)

595 temperature of incubation and (c) light of incubation on germination recovery percentage (mean

596 ± S.E.) of Salsola drummondii seeds.

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Fig. 1.

a) Maternal salinity

(b) Temperature

(c) Light

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Fig. 2.

(a) Maternal salinity

(b) Temperature

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Fig. 3.

(a) Maternal salinity

(b) Temperature

(c) Light

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