Introduction
Plants in hot desert regions are exposed to abiotic extremes, where high solar radiation leads to high temperatures, spatial and temporal unpredictability of precipitation (Noy-Meir, Reference Noy-Meir1973; Merquiol et al., Reference Merquiol, Pneuli, Cohen, Simovitch, Rachmilevitch, Goloubinoff, Kaplan and Mittler2002) and, often, saline conditions (Rewald et al., Reference Rewald, Eppel, Shelef, Hill, Degu, Friedjung, Rachmilevitch and Bell2011). While this combination of stress factors may be harder to cope with than each factor by itself, salinity and aridity act, to some extent, in opposite directions, due to the hygroscopic nature of salt and reduced evaporation with increased salinity (Al-Shammiri, Reference Al-Shammiri2002). Thus, saline soils often have higher water content than less saline soils, and therefore plant responses to a combination of drought and salt stress are complex and can even be synergistically advantageous (Fu et al., Reference Fu, Li and Chen2012).
Most plants are not armed with mechanisms to utilize saline water, and suffer from hydrological drought due to osmotic constraints. Halophytes are the exception. These plants have evolved a variety of adaptations to compensate for osmotic stress, affording survival in saline environments (Waisel, Reference Waisel1972). Once they are established in the soil, halophytes can withstand extreme salinities of more than 200 mM NaCl (Flowers and Colmer, Reference Flowers and Colmer2008). Their unique adaptations to saline environments have recently attracted more attention (Flowers and Colmer, Reference Flowers and Colmer2015) and hold potential for agricultural development (Ventura et al., Reference Ventura, Eshel, Pasternak and Sagi2015). Waisel (Reference Waisel1972) suggested dividing halophytes into two categories: plants that avoid salt (facultative halophytes) and plants that require salt for survival or better development (obligatory halophytes). Obligatory halophytes not only tolerate high levels of salinity but also reach optimal levels of growth under saline conditions (Flowers et al., Reference Flowers, Troke and Yeo1977; Ungar, Reference Ungar1991; Shelef et al., Reference Shelef, Lazarovitch, Rewald, Golan-Goldhirsh and Rachmilevitch2010).
Coping with aridity and salinity in the initial growth stages is difficult, even for halophytes. The most vulnerable stage in a dry environment is when wet seeds are germinating and developing into seedlings: dormancy is no longer protective, and the roots have not yet developed sufficiently to supply adequate quantities of water and nutrients. Young seedlings in hot deserts are often exposed to desiccation after germination. The success of halophyte populations is greatly dependent on their seeds’ germination response to salinity (Tobe et al., Reference Tobe, Li and Omasa2000; Song et al., Reference Song, Feng, Tian and Zhang2005). Halophytes can maintain seed viability for extended periods under hypersaline conditions and then initiate germination when the salinity stress is reduced (Woodell, Reference Woodell1985; Khan and Ungar, Reference Khan and Ungar1998).
The ability of seeds to remain viable for extended periods of salinity has been investigated (Naidoo and Naicker, Reference Naidoo and Naicker1992; Keiffer and Ungar, Reference Keiffer, Ungar and Ungar1995). Seeds typically germinate early in the spring when soil salinity levels are reduced, prior to the period of highest salt stress (Ungar, Reference Ungar1991). By the end of the summer, the maximum yearly concentration of salt on the soil crust can reach levels of about 8% (Waisel, Reference Waisel1972) or more. Rainfall can quickly leach salts from the soil surface and supply water for the seeds. Thus, for successful establishment of plants in saline environments, their seeds must remain viable under high salinity and germinate when salinity decreases (Khan and Ungar, Reference Khan and Ungar1997).
The ability of young seedlings to tolerate periods of drought between precipitation events represents an important survival strategy, particularly under extreme desert conditions with unpredictable amounts and distribution of rainfall (Evenari, 1982; Gutterman, Reference Gutterman2002). Species with young seedlings that are able to survive fairly long periods of desiccation are termed ‘resurrection plants’ (Farrant, Reference Farrant2000; Wagner et al., Reference Wagner, Schneider, Mimietz, Wistuba, Rokitta, Krohne, Haase and Zimmermann2000) and examples of these are: Psammochloa villosa (Trinius) Bor (Poaceae) in the Ordos desert in China (Huang et al., Reference Huang, Dong and Gutterman2004) and Leymus racemosus (Lam.) Tzvelev (Poaceae) (wild rye) in sand dunes in China (Huang and Gutterman, Reference Huang and Gutterman2004). Seedlings of Schismus arabicus Nees (Poaceae) can potentially survive over 8 weeks of desiccation (Gutterman et al., Reference Gutterman, Gendler and Rachmilevitch2010).
We used B. indica as our focal species. This species is widely distributed in the Negev desert and has been shown to have unique halophytic adaptations, such as ‘positive halotropism’, the growth of roots toward an optimum soil salinity (Shelef et al., Reference Shelef, Lazarovitch, Rewald, Golan-Goldhirsh and Rachmilevitch2010). We have studied B. indica previously (Shelef et al., Reference Shelef, Lazarovitch, Rewald, Golan-Goldhirsh and Rachmilevitch2010, Reference Shelef, Gross and Rachmilevitch2012, Reference Shelef, Gross and Rachmilevitch2013; Freedman et al., Reference Freedman, Gross, Shelef, Rachmilevitch and Arnon2014) and, therefore, we were interested in aspects of its tolerance to salinity and drought. The Bassia genus is widespread in the Mediterranean and in eastern Asia. Our current goal was to study the salinity tolerance of B. indica in its early growth stages. In addition, we compared B. indica to two other Bassia species that originate from a different hot desert. Similar to other members of Amaranthaceae and closely related Chenopodiaceae, Bassia species produce small seeds that are dispersed by the wind and therefore have little storage tissue. This exposes the seeds to a rather vulnerable stage in their initial phases of growth. Seeds of the three Bassia species were tested for their germination success and seedling survival under desiccation. In their natural habitats, these three species are exposed to short rain events followed by periods of drought. Wet soil engenders seed germination, whereas dry periods dry the young seedlings. These arid conditions are often accompanied by saline conditions.
We asked how dry conditions and salinity would affect germination, initial growth and desiccation tolerance of these halophytes in their early growth stages. Our overall goal was to study how low water availability and salinity affect seedling viability of B. indica. We hypothesized first that these three species would exhibit adaptations to water shortages within 24 h of germination, resulting in rapid germination, root elongation and high rates of seedling survival in the face of desiccation. Second, we hypothesized that for obligatory halophytes, such as B. indica (Shelef et al., Reference Shelef, Lazarovitch, Rewald, Golan-Goldhirsh and Rachmilevitch2010), salinity would have a positive effect on germination success and desiccation tolerance.
Materials and methods
Study sites
This study compares three plant species of the same genus, but from rather distant origins: the Kyzyl Kum desert in central Asia and the Negev desert in Israel. The Kyzyl Kum desert is a large, hot desert located between the Amu Darya and Syr Darya rivers, mainly in Uzbekistan. Mean annual precipitation is 113 mm. Salty pans (solonchaks) are typical for the region (Kozhoridze et al., Reference Kozhoridze, Orlovsky and Orlovsky2012). These soils are characterized by wet upper soils and temporal waterlogging (Gintzburger et al., Reference Gintzburger, Toderich, Mardonov and Mahmudov2003) . Along the Amu Daria River solonchaks are often poor in calcium content and rich in sodium chloride (NaCl) and magnesium (Gintzburger et al., Reference Gintzburger, Toderich, Mardonov and Mahmudov2003). The area has suffered accelerated desertification, characterized by sinking groundwater levels and land salinization (Micklin, Reference Micklin2007).
The Negev is a subtropical desert, the northernmost edge of the global desert belt, which extends into Israel. Winter is characterized by 100–250 mm of rain (annual average). The wet winter is the growth time of most plants, perennials dominated by shrubs and annuals. Loess soil predominates in the area that B. indica occupies. Loess is an aeolian soil formed from desert dusty material. It is flat and deep, low in organic matter, moderately alkaline and the dominant cation is calcium (Ravikovitch, Reference Ravikovitch1960).
Plant species
B. indica (Wight) A.J. Scott is an annual that grows in the Negev desert. Its growth is limited during the winter, following accelerated development during the summer. B. indica shrubs inhabit dry, saline desert areas in the Judean and Negev deserts where they are considered an excellent pasture herb (Youssef et al., Reference Youssef, Fahmy, Abeer and El Shaer2009). Bassia species may be used as animal fodder, and B. indica has been proposed as a hyper salt accumulator for salt phytoremediation in constructed wetlands (Shelef et al., Reference Shelef, Gross and Rachmilevitch2012, Reference Shelef, Gross and Rachmilevitch2013; Freedman et al., Reference Freedman, Gross, Shelef, Rachmilevitch and Arnon2014).
We compared B. indica to two other species of the genus Bassia, family Amaranthaceae. All studied species possess small, wind-disseminated seeds. B. iranica (L.) A.J. Scott is a perennial shrub that germinates during the winter. B. prostrata (L.) A.J. Scott is an annual, germinating in March–April (Gintzburger, 2003). B. iranica and B. prostata are common in the Kyzyl Kum desert. These species populate periodical salt marshes on alkaline solonchak soils, or sandy soils and dunes (Lobova, Reference Lobova1960). They are commonly used in pasture and fodder production, and local inhabitants use their trunks as wood fuel.
Seed collection
Mature seeds of the three Bassia species were collected from natural populations. B. indica seeds were collected in the winter of 2009 from the Sede Zin plateau near the Sede Boker campus of Ben Gurion University in the Negev desert. Experiments on B. indica were conducted in the winter of 2009. B. iranica and B. prostrata seeds were collected in October 2004, in the periodical saline swamps of the Kyzyl Kum desert in Uzbekistan, Mujnak region, 20 km south of the Aral Sea. These seeds were kept in dry storage at the Sede Boker campus at room temperature for 6 months and germination experiments were carried out in May 2005.
Germination in saline solutions
Seeds were placed in 50-mm-diameter Petri dishes on a Whatman no. 1 filter paper with 3 ml distilled water under ambient conditions [25°C, 17% relative humidity (RH)], measured by thermo-hydrograph (Wilhelm Lambrecht Gmbh, Göttingen, Germany). Four replicates of 25 seeds each for each species and solution were used for statistical analysis. Seeds in each replicate were wetted with 3 ml of the following solutions: fresh water (~0 mM), 1% NaCl (~170 mM), 2% NaCl (~350 mM) and 3% NaCl (~500 mM). Germination was recorded 24 h after wetting and germination percentages were calculated. We waited for an additional 72 h to make sure germination was not delayed.
Dynamics of seedling desiccation
Five groups of ten dry seeds from each of the three Bassia species were used as replicates. Seeds were weighed in each replicate before wetting with different saline solutions as mentioned above. After 24 h of wetting in light (24°C), seedlings appeared and were weighed. The young seedlings were then transferred to a dry filter paper and dried for a week (20–25°C; 10–15% RH). Young seedlings developed roots quickly (see supplementary Movie S1). The dry seedlings were weighed 24 h after desiccation had commenced, and then every 24 h until weight loss ceased, 96 h after the beginning of desiccation. No further decrease in weight occurred in the subsequent 48 h and therefore results are given for four time points: phase 1, dry seeds; phase 2, 24 h after wetting, desiccation started immediately after this phase; phase 3, 24 h after the beginning of desiccation; and phase 4, 96 h after the start of desiccation.
Seedling survival after desiccation
Following wetting and desiccation, as described in the previous section, seeds were kept in dry storage for four periods of time: (1) 7 d; (2) 14 d; (3) 21 d; and (4) 30 d or more. Twenty-five seeds were used in each replicate. Time of desiccation was selected to simulate the time seedlings spend in the field after germination and before the subsequent rain event that allows root establishment. Temperatures were measured daily by a minimum–maximum thermometer and 10–15% RH was maintained. At the end of the desiccation period, the dry seedlings were rewetted. Each group of seedlings was rewetted in the solution originally used for germination. Surviving seedlings were counted 48 h after rehydration, according to their ability to resume growth. Seedling survival was calculated as the percentage of live seedlings out of the total number of seedlings in a Petri dish. Results of the different desiccation times were pooled. Different desiccation times were only relevant for comparison within B. indica seedlings, because of the high mortality rate of the other species. Root elongation was determined as the average of 13 seedlings (out of a maximum of 25 if all survived). The minimal surviving rate and threshold, determined accordingly following the same desiccation procedure, was 13.
Data analysis
Statistical analyses were conducted with STATISTICA version 10.0 software (StatSoft Inc., Tulsa, Oklahoma, USA). Means and standard errors were calculated for each set of repetitions, and these numbers (n, averages and SE) are specified in the Results section. The probability of a fit to a normal distribution was determined by the Shapiro–Wilk test prior to the statistical test. Most data deviated from a Gaussian distribution and therefore were compared by non-parametric methods. Kruskal–Wallis analysis and Wilcoxon to locate the differences or Mann–Whitney U tests were performed. P ≤ 0.05 was considered to be statistically significant.
Results
Seedling growth and desiccation
Seedlings of all three Bassia species germinated rapidly, within 24 h after wetting. B. indica seeds accumulated water to 4.48 times their initial weight, significantly more than B. iranica and B. prostrata, which accumulated 2.5 and 2.02 times their initial weight, respectively (Table 1). There was a gradual decrease in seedling weight over 96 h of desiccation, with the highest loss for B. indica (Table 1). B. indica seedlings showed the most rapid water accumulation (i.e. high Δi weight, Table 1) and desiccation (i.e. total water loss within 24 h).
Dynamics of seedling desiccation in Bassia species. Δ weight is the difference (%) between the former phase and the current as indicated hereinafter. Δi weight is gain of weight during wetting, from initial dormant phase i to phase 1; Δ1 weight is the difference between seedling weight 24 h after germination (phase 2) and their fresh weight after wetting (phase 1); Δ2 weight is the difference between seedling weight 96 h after germination (phase 3) and their fresh weight after wetting (phase 1)
Germination success under salinity
Germination success of B. indica did not decline significantly with increasing salinity in the growth solution up to 2% NaCl. Moreover, only B. indica seeds germinated successfully in growth solution with 3% NaCl (Fig. 1 and supplementary Movie S1), although germination was nearly 60% lower than in 2% NaCl (30.75 ± 13.8% vs. 86.5 ± 5.6%, respectively; Kruskal–Wallis test, P= 0.02, n= 4) (Fig. 1). Seeds of B. iranica and B. prostrata germinated well in fresh water, with no dormant period. For both species, germination decreased with increasing NaCl concentrations. Germination success of B. iranica seeds dropped significantly from 99.5 ± 0.5% in fresh water to 56 ± 5.6% in growth solution with 2% NaCl (Kruskal–Wallis test, P= 0.005, n= 4). No germination of B. prostrata and B. iranica was observed in 3% NaCl.
Germination of Bassia species subjected to different extents of salinity. Bars represent mean ± SE of germination success for each treatment. Different letters denote significant differences between treatments within B. indica (a, b) or B. iranica (x, y); within B. prostrata, treatments were not significantly different at P < 0.05 (n= 8).
Seedling survival after desiccation
Root elongation after a period of 14–30 d of desiccation was very rapid upon rewetting, with root lengths reaching up to a maximum of 40 mm within the first 48 h. Roots of B. indica were significantly longer when seedlings were rewetted with fresh water after 7 and 21 d (Fig. 2). In B. indica, seedling survival after desiccation was positively correlated with increasing salinity up to 2% NaCl, an increase of nearly 83%, from 17.25 ± 9.6% in fresh water to 100% in 2% NaCl (Fig. 3). Moreover, B. indica seedlings remained viable after desiccation and rewetting in 3% NaCl, with 62.5 ± 18.3% seedling survival (Fig. 3). However, following 30 d of desiccation after the initial wetting, roots developed to significantly greater lengths in 2% NaCl as compared to fresh water.
Bassia indica root growth in different saline solutions after a desiccation period. Different letters denote significant differences between treatments within a drought period at P < 0.05 (n= 8).
Effect of salinity on Bassia seedling survival following desiccation. Bars represent mean ± SE of seedling survival for each treatment. Different letters denote significant differences between treatments within B. indica at P < 0.05 (n= 8 for each species and solution).
Effect of desiccation period and salinity on seedling success of B. indica
When exposed to relatively low salinities (fresh water and 1% NaCl), seedlings of B. indica seemed to exhibit decreased survival success with longer desiccation periods before rewetting (7 d desiccation as compared to 21 d or more) (Fig. 4). However, the apparent difference in seedling survival following short vs. long periods of desiccation was not statistically significant (Mann–Whitney U test, P > 0.05). The apparent increased survival at higher salinities after short desiccation (7 d) was insignificant as well (Kruskal–Wallis test, P > 0.05) (Fig. 4). Following 21 d of desiccation, seedling survival in 2% NaCl (100%) was 82.7% higher than that in fresh water (17.25 ± 9.6%, Kruskal–Wallis test, P < 0.002, n= 4). These seeds also showed high survival rates (62.5 ± 18.3%) in 3% NaCl (Fig. 4).
Effect of desiccation period on survival of Bassia indica seedlings in different salinities. Bars represent mean ± SE for each treatment. Different letters denote significant differences between treatments within the same period of desiccation after germination: 7 d or more than 21 (21+) d, at P < 0.05 (n= 4 for 7 d, n= 8 for 21+ d).
Discussion
Seedling growth in an arid environment
Growth dynamics showed adaptations to water shortage within 24 h after germination. All three species exhibited rapid germination and root elongation (Fig. 2) and relatively high germination rates (a maximum of over 80%, Fig. 1). B. iranica and B. indica showed relatively high seedling survival rate (over 80%) in 2% NaCl (Fig. 3). Rapid germination and seedling development is characteristic of the Chenopodiaceae under dry desert conditions.
Growth media have a great influence on root growth. However, the root dynamics reported here highlight the rapid growth potential of Bassia species compared with other species under similar conditions (Wallace et al., Reference Wallace, Rhods and Frolich1968). Rapid germination and root extension are common adaptations among inhabitants of hot deserts (Leon et al., Reference Leon, Squeo, Gutierrez and Holmgren2011); for example, Salsola kali L. (Amaranthaceae) germinates 29 min after wetting (Wallace et al., Reference Wallace, Rhods and Frolich1968), and Schismus arabicus after 4 h (Gutterman, Reference Gutterman2001).
Effect of salinity on germination and seedling survival
Salinity had a negative or no effect on the germination rates of B. iranica, B. prostrata and B. indica; the latter being the only species able to germinate in 3% NaCl (Fig. 1). On the other hand, salinity had a positive effect on B. indica seedling survival after a long desiccation period (Figs 2 and 3), although it had no significant effect on B. prostrata or B. iranica. This might be explained by different coping strategies. Previous studies have shown that B. indica behaves as an obligatory halophyte, requiring optimal salinity that is higher than in fresh water (Shelef et al., Reference Shelef, Lazarovitch, Rewald, Golan-Goldhirsh and Rachmilevitch2010). However, our germination experiments did not show that B. indica seeds favour salinity over fresh water. In this regard, B. indica did not behave as an obligate halophyte. On the other hand, salinity increased B. indica seedling elongation and survival after long periods of desiccation (Figs 3 and 4). With its ability to compensate for osmotic stress, combined with a saline environment, B. indica benefited from saline growth media. This result supports studies reporting B. indica as an obligatory halophyte with resistance to a wide range of salinities (Shelef et al., Reference Shelef, Lazarovitch, Rewald, Golan-Goldhirsh and Rachmilevitch2010, Reference Shelef, Gross and Rachmilevitch2012, Reference Shelef, Gross and Rachmilevitch2013). To date, only a few plants from dry saline deserts have been found to have the ‘resurrection’ adaptation of seedling desiccation tolerance (Friedman et al., Reference Friedman, Stein and Rushkin1981; Farrant, Reference Farrant2000; Wagner et al., Reference Wagner, Schneider, Mimietz, Wistuba, Rokitta, Krohne, Haase and Zimmermann2000; Gutterman, Reference Gutterman2002). As far as we know, there are no previous publications presenting survival of seedlings after desiccation and rewetting with solutions of up to 3% NaCl. The ability to germinate, survive and renew elongation in up to 3% NaCl, even after long periods of desiccation, among plant seedlings has not been reported. It is an important survival strategy for species that inhabit saline deserts with unpredictable precipitation amounts and distribution.
Many of the world's arid and semi-arid regions consist of soils and water resources that are too saline for most common economic crops (Nerd and Pasternak, Reference Nerd and Pasternak1992). An important economic solution in such areas is the utilization of halophytic plants for pasture and fodder (Yeo and Flowers, Reference Yeo and Flowers1980). The use of halophytes can thus contribute to reduction of grazing pressure, and also to land restoration to combat desertification.
We quantified some positive effects of salinity on halophytes’ tolerance to aridity. Our results show that salinity can protect halophyte seedlings from desiccation, when it is present within 24 h after germination. Soil salinity is strongly correlated with moisture (Eisenberg et al., Reference Eisenberg, Dan and Koyumdjisky1982) and our findings reveal the impact of these two combined abiotic stress conditions on seedling viability of halophytes. These findings may contribute to a better understanding of survival mechanisms of plants under harsh desert conditions with periodical fluctuations in aridity and soil salinity.
Supplementary material
Movie S1. A movie of Bassia indica germination. Seeds were germinated in fresh water, 1%, 2% or 3% NaCl. The movie was created as a stop-motion track of frames taken at 5-min intervals for 90 h after initial wetting. Constant neon lighting was required to enable the photography.
To view supplementary material for this article, please visit http://dx.doi.org/10.1017/S0960258515000409.
Financial support
The study was partially supported by Israel Science Foundation (ISF) number 958/10 and by the Rosenzweig–Coopersmith Foundation (RCF).
Conflicts of interest
None.
