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Shoots of Thellungiella derived by micropropagation were used to estimate the plants' salt tolerance and ability to regulate Na+ uptake. Two species with differing salt tolerances were studied: Thellungiella salsuginea (halophilla), which is less tolerant, and Thellungiella botschantzevii, which is more tolerant. Although the shoots of neither ecotype survived at 700 mM NaCl or 200 mM Na2SO4, micropropagated shoots of T. botschantzevii were more tolerant to Na2SO4 (10–100 mM) and NaCl (100–300 mM). In the absence of roots, Na2SO4 salinity reduced shoot growth more dramatically than NaCl salinity. Plantlets of both species were able to adapt to salt stress even when they did not form roots. First, there was no significant correlation between Na+ accumulation in shoots and Na+ concentration in the growth media. Second, K+ concentrations in the shoots exposed to different salt concentrations were maintained at equivalent levels to control plants grown in medium without NaCl or Na2SO4. These results suggest that isolated shoots of Thellungiella possess their own mechanisms for enabling salt tolerance, which contribute to salt tolerance in intact plants.
Comparing closely related glycophytes, especially those whose genomes have been cloned, is a valuable strategy for studying the specific biochemical and physiological features of native halophytes. The genome of Thellungiella salsuginea (halophila) has high homology to the genome of Arabidopsis and has become popular in the study of plant tolerance to environmental stresses. Similar to Arabidopsis, T. salsuginea has a short life cycle, produces large quantities of seed, is a self-pollinator and can be easily transformed by plunging inflorescences into Agrobacterium suspensions.1 T. salsuginea can grow in areas with high soil salinity and at low temperatures, making it a useful species for studying these stresses simultaneously or separately.2
Salt stress induces Na+ accumulation in shoots of T. salsuginea (ecotype Shandong) at significantly lower levels than for Arabidopsis thaliana.3 Studies with Na+ radioisotopes found that the root is the main barrier to Na+ uptake in T. salsuginea, which indicates that the root system is the main part of the plant that mediates salt tolerance in this species.4
Despite recent studies into the mechanism of salt tolerance in T. salsuginea, many issues remain unclear, including the role of the shoot. To elucidate the contribution of the shoot to salt tolerance of Thellungiella spp. plants, we examined the growth and development of isolated shoots derived by micropropagation. Natural variability within the genus Thellungiella allowed us to compare two Thellungiella species that differ in salt tolerance; T. salsuginea (halophilla) is less tolerant and T. botschantzevii is more tolerant. These species were collected from different climatic zones diverging in temperature regime, day length and soil salinity. We developed a protocol for isolating shoots in vitro by microclonal propagation, established the range of NaCl and Na2SO4 concentrations where shoots could develop roots and where they could survive on their own, and finally estimated the ability of the shoots to maintain or accumulate Na+ and K+.
Sterile plants of two species of Thellungiella were grown from micropropagation-derived shoots. These plants were very similar to plants grown from seed (Fig. 1). Shoots of T. botschantzevii had well-developed rosettes and greater biomass than those of T. salsuginea (Table 1). Growing plants of both species in 100 mM NaCl led to decreased shoot biomass compared to controls. Further increases in NaCl concentration (200 mM and 300 mM) did not significantly affect biomass. The biomasses of T. salsuginea and T. botschantzevii shoots grown in 600 mM were reduced by 73% and 88%, respectively, compared to controls. A concentration of 700 mM NaCl was lethal for shoots of both species.
Addition of 10 mM and 25 mM Na2SO4 to the medium did not affect the growth of T. salsuginea plantlets, but slightly increased biomass accumulation of T. botschantzevii plantlets. Higher Na2SO4 concentrations in the medium inhibited biomass accumulation in both species, although the T. salsuginea was affected to a greater degree. A concentration of 200 mM Na2SO4 was lethal for both species. The difference in the tolerance of T. botschantzevii and T. salsuginea to Na2SO4 salinity could be related to higher sulphate concentrations found in soil from the site where the T. botschantzevii was collected.
In addition to changes in shoot biomass, salt stress also affected rhizogenesis. Inhibition of rhizogenesis was observed earlier than statistically different changes in biomass accumulation at several concentrations (Table 1). Isolated shoots of T. salsuginea did not form roots at 300 mM or 500 mM NaCl; however, biomass was reduced only by 16% and 20% compared to plants grown at 200 mM NaCl, which did form roots. Moreover, the shoot biomass of T. botschantzevii plantlets grown at 200 mM and 300 mM was 9% and 5% less, respectively, than in plants grown at 100 mM, which formed roots (Table 1).
For plants without roots, salt stress caused by high concentrations of Na2SO4 was more toxic than high concentrations of NaCl. Plants of T. salsuginea without roots accumulated 30% less biomass at 50 mM Na2SO4 than at 25 mM Na2SO4, where roots were present. T. botschantzevii plants grown in the same concentrations of Na2SO4 (50 mM, with weak rhizogenesis, and 25 mM) showed a 29% reduction in biomass.
We used regression analysis to estimate the influence of rhizogenesis and salt concentration on plant biomass. Table 2 shows that there was a high correlation between variables, as most values for coefficients were higher than 0.9. Based on these coefficients, salt concentration had the greatest effect, whereas rhizogenesis had less of an effect on plant biomass. Moreover, this trend was found for both species and both types of salinity. The correlation between plant biomass and rhizogenesis was R = 0.751 for T. salsuginea and R = 0.805 for T. botschantzevii. However, the combined effect of both variables on plant biomass was higher in T. botschantzevii than in T. salsuginea for both types of salinity.
Under both salt stresses, isolated shoots accumulated Na+ at similar levels, but maintained K+ at control levels (Fig. 2A and B). However, the increase in Na+ in the growth medium did not cause a proportional increase of Na+ accumulation in shoots, even when the plants did not form roots.
These results demonstrate that isolated shoots of Thellungiella possess mechanisms for adapting to salt stress, as the lack of root growth at certain applied concentrations of NaCl and Na2SO4 did not affect shoot biomass. The correlation coefficient R, calculated to assess the interaction between shoot biomass and rhizogenesis (Table 2), was higher in plants exposed to Na2SO4 salt stress than to NaCl. This may indicate a higher level of interaction between roots and shoots in plants' adaptation to Na2SO4 salt stress. To our knowledge, there is no information on how sulphur-containing compounds might affect shoot-root interaction, but there are some indications that these compounds are involved in regulating long distance transport of mineral ions and other molecules in plants. Genes encoding sulphate transporters are expressed in the phloem,7 glutathione quickly exchanges between phloem and xylem8 and is involved in rhizogenesis,9 and thiol-containing glutamylcysteine synthase is transported to roots.10 In contrast, NaCl-induced salt stress leads to a reduction in sulphur-containing compounds in plants, including halophytes,11 which could also account for the weaker interaction between shoots and roots found here (Table 2).
Na+ accumulation in the shoots of both Thellungiella species demonstrated that shoots themselves could regulate Na+ uptake. We did not observe a proportional increase of Na+ accumulation in shoots when the Na+ concentration in the medium was increased. Doubling the Na2SO4 concentration in media (from 50 mM to 100 mM) led to a 1.5-fold increase in Na+ in shoots, and a three-fold increase in NaCl concentration in the medium (from 100 to 300 mM) led to a 1.8-fold increase in the Na+ concentration in shoots. The regulation of Na+ uptake in shoots may be due to the activity of a plasma membrane Na+/H+ antiporter, which excludes Na+ to the apoplast,12 and/or to the shoot's ability to transport Na+ back to the medium via cycling in the phloem.13
Interestingly, these two species did not differ in terms of Na+ accumulation in the shoots, but did differ in the reduction of shoot biomass under salt stress. Comparison of these two species demonstrated that, in this genus, there is no direct link between Na+ accumulation in shoots and shoot biomass under salt stress.
Another interesting feature of the isolated shoots was their ability to maintain normal K+ levels under salt stress. The potassium levels in isolated T. botschantzevii and T. salsuginea shoots differed from the results measured from whole Thellungiella (Shandong ecotype) plants, where a reduction of K+ concentration in shoots was previously observed.2 This discrepancy can be explained by the different conditions of cultivation, including different media. However, it may be that the main difference between these two observations is caused by different methods of approach: isolated shoots versus whole plants.2 Hence, it can be suggested that the K+ decrease in shoots under salt stress is linked to processes in the roots. For example, the root may not be able to maintain K+ uptake in the presence of increased Na+ concentrations in the cytosol, as was reported for Arabidopsis, which is a close relative to Thellungiella spp.14
Therefore, isolated shoots from the two ecotypes of Thellungiella can survive salt stress induced by NaCl and Na2SO4, even when roots are not formed. In addition, they can maintain K+ concentrations in shoots at control levels. Our results suggest that isolated shoots of Thellungiella possess their own mechanism of salt tolerance, which contributes to the salt tolerance of the intact plant. Similarly, recent studies on isolated leaves of lucerne demonstrated that they could resist salt stress,15 and the shoots of many other plant species also exhibit such resistance.16 To determine the molecular mechanisms that are responsible for shoot salt resistance, further studies on the cellular and the whole plant level are needed.
Seeds of two species of Thellungiella were collected from two locations in the Russian Federation: T. botschantzevii from the Saratov Region (51°N; 45–46°E) and T. salsuginea from Yakutsk, in the Sakha Republic (61°N; 130°E).5,6 Chemical analysis of soil samples from the sites where seeds were collected demonstrated a prevalence of SO42− over Cl−, and Ca2+ and Mg2+ over Na+; the pH was 7.6. The concentration of soluble salts was 2.10% for the Saratov Region site and 0.62% for the Sakha Republic site. T. botschantzevii plants, which grow under higher salt salinity in natural environments, were also found to be more tolerant to NaCl in glass house experiments (data not shown).
Seeds of Thellungiella were surface-sterilized in 0.1% diacid (two parts 0.667 g/L N-cetylpyridinium chloride and one part 0.333 g/L mercury chloride dissolved in ethanol) for 7 min, then rinsed in sterile water three times. After drying, the seeds were placed onto S MS agar media with 2% sucrose (w/v) in tubes. After 25–30 days, shoots and roots were removed from the growing plants and were placed onto full MS agar media with vitamins, 2% sucrose, 0.025 mg/L BAP and 0.1 mg/L IMA. Tubes with seeds were placed a Fitotron 600H plant growth chamber (Weiss-Gallenkamp, Loughborough, UK) with radiance at 250 µmol m−2 s−1 during the 16 h photoperiod, and temperatures of 25°C/18°C (day/night).
After 25–30 days of growth, the shoots formed 4–7 adventitious shoots, which were separated and placed onto fresh media. After three passages, 1.5 cm shoots with the roots removed were placed onto S MS agar with 2% sucrose and 0.1 mg/L IMA as a control. Media for salt treatments were the same as the control with the addition of 100, 200, 300, 500, 600 or 700 mM NaCl or 10, 25, 50, 75, 100 or 200 mM Na2SO4. Plants were harvested 30 days after exposure to salts. The effect of salt stress was assessed by analysing the fresh biomass of shoots, the number of roots formed and Na+ and K+ contents. Shoots were dried at 70°C and digested in 0.1 M HNO3.2 Na+ and K+ concentrations were determined by atomic absorption using a Kvant 2A spectrophotometer (Kortek, Russia).
All experiments were repeated three times and 10–12 plants were assessed each time. Results are presented as mean ± S.E. To analyse the relationship between root formation and shoot biomass, we used the regression analysis tool from Microsoft Office Excel 2003. Multivariable correlation coefficients were calculated using the built-in data analysis tool in MS Excel 2003.
The multiple correlation coefficient (R) was used to estimate the relationship between biomass, rhizogenesis and salt concentration. This coefficient estimates the combined influence of two or more variables on the observed (dependent) variable, and can vary from −1 (perfect negative correlation) through 0 (no correlation) to +1 (perfect positive correlation). In the current study, R estimated the influence of rhizogenesis and salt concentration on plant biomass.
This work was partially supported by grant NWO-RFBR 05-04-89005.
Previously published online: www.landesbioscience.com/journals/psb/article/9799