Introduction

Salt stress negatively influences agricultural yield throughout the world, whether it is for subsistence or economic gain (Flowers 2004). Soil salinity is one of the most important factors that limit crop production in arid and semi-arid regions. The United Nations Environment Program estimated approximately 20% of agricultural land and 50% of cropland in the world is salt-stressed (Flowers and YEO 1995). Iran is one of the countries that suffer from severe salinity problems and 10% of Iran’s land is affected by salt (Saboora et al. 2006).

The plant response to salinity consists of numerous processes that must function in coordination to alleviate both cellular hyper salinity and ion disequilibrium that produce a secondary effect (Hasegava et al. 2000). Plants are either dormant during the salt episode, or they make cellular adjustment to tolerate the saline environment (Yokoi et al. 2002). Addition of NaCl to the nutrient solution causes a marked decrease in growth (Sepehr et al. 2003). Several factors may contribute to the reduction in growth exhibited by plants under salinity stress. One significant factor may be related to the inhibition of vascular tissue production under stress (Sepehr et al. 2003). Salinity inhibits plant growth mainly by water deficit, ion toxicity, and ion imbalance (Greenway and Munns 1980).

Wheat is the source of almost 20% of total necessary calories for the world’s population (Perviaz et al. 2002). In wheat, genotypic variation in salt-tolerant has been associated with low rate of Na+ uptake and transport, and high selectivity for K+ or Ca++ over Na+ (Schachtman and Munns 1992). Plants absorb the constituent ions of a saline substrate in varying degrees. This may result in harmful accumulations of particular ions or decreases in the absorption of some essential nutrients, in addition to the depressive effect on the activity of the endogenous growth hormones (Dawh et al. 1998). Salt tolerance in the Triticeae is associated with sodium exclusion (El-Hendway et al. 2005). The presence of salt in soil solution decreases the osmotic potential of soil, thereby resulting in water stress and making it difficult for plant to absorb the water necessary for growth (El-Hendway et al. 2005). Generally, plants are able to tolerate salinity by reducing leaf osmotic potential via the synthesis of organic solutes or the accumulation of inorganic ions (Hasegava et al. 2000).

Attempts have been made to overcome the adverse effect of salinity; one of them is application of growth regulators (Abou El-Khashab et al. 1997; Kaur et al. 1998; Özmen et al. 2003). A more efficient group of compounds, the triazoles, was developed to act on ent-kaurene oxides in the GA biosynthetic pathway. It has been demonstrated that the triazoles protect plants against various stresses including drought, low and high temperatures, UV light and air pollution. They have been referred to as plant “multi-protectants” (Fletcher et al. 2000). Paclobutrazol (PBZ) [(2RS, 3RS)-1-(4-chlorophenyl)-4, 4-dimethyl-2-(1, 2, 4-triazol)-pentan-3-ol] is a triazole plant growth regulator that consists of two enantiomers, namely 2R, 3R and 2S, 3S forms (Hedden and Graebe 1985). Triazoles interfere with three steps of ent-kaurene oxidation pathway and inhibit the formation of ent-kaurenol, ent-kaurenal, and ent-kaurenoic acid (Izumi et al. 1985). These microsomal oxidation reactions are catalyzed by kaurene oxidase and interference with the different isoforms of this enzyme could lead to inhibition of gibberellins (GA) biosynthesis and abscisic acid (ABA) catabolism (Rademacher 1997). Thus, PBZ blocks the biosynthesis of the active GA and therefore decreases plant growth and development (Mehouachi et al. 1996). The triazole that mediates stress protection often explained in terms of hormonal changes such as an increase in cytokinins, a transient rise in ABA and a decrease in ethylene (Asar-Boamah and Fletcher 1986; Fletcher and Hofstra 1988; Mackay et al. 1990). Some researchers indicated that PBZ reduced the effect of salt stress on guava and grapes (Abou E1-Khashab 1991; Eliady et al. 1992; Salama et al. 1992). The most pronounced effect of triazole on plants is a reduction in height (Fletcher and Hofstra 1988). It was hypothesized that PBZ-treated plants had a better quality of growth under salt stress than non-treated plants, due to the slower growth rate of the former (Abou El- Khashab et al. 1997).

Therefore, the primary objective of the present study was to determine whether exogenously applied PBZ could alleviate the salt stress in genetically diverse wheat cultivars. For this purpose, we compared the response of both salt-tolerant and salt-sensitive cultivars of wheat to salinity and PBZ application.

Materials and methods

Plant culture and treatment

Seeds of a salt-tolerant (Karchia-65) and salt-sensitive (Ghods) cultivar of wheat (Triticum aestivum) (Pustini 2001) were obtained from the Seed and Improvement Institute at Karaj, Iran. The experiment was conducted at Alzahra University, Tehran, Iran in November 2005. The seeds were cultured in 1.5 l pots, filled with vermiculite in a greenhouse with supplementary light to extend the photoperiod to 16 h per day (Berova et al. 2002; Kerepsi and Galiba 2000; Sepehr et al. 2003). Air temperature ranged from 22°C to 26°C during the day and 15–18°C during the night (Erdei et al. 2002). Humidity ranged from 40% to 60%. Ten seeds were sown in each pot. The plants were irrigated every 4 days and fertilized once a week by the standard Hoagland nutrient solution (pH 6.0). Ten days after sowing, the seedlings were thinned to four similar plants per pot. The experiment was designed as a completely randomized block in a split plot with the PBZ treatment as main plot, cultivar as sub-plot and NaCl treatment as sub-sub-plot (Crandall et al. 2004; Klein et al. 2002). Each main plot consisted of 32 plots and there were four replications per NaCl treatment. At two-leaf-stage (Klein et al. 2002), the plants were sprayed with 0 (distilled water) and 30 ppm of PBZ solution (Dawh et al. 1998). PBZ was diluted in distilled water and the solution was applied uniformly to the plants as a fine spray using an atomizer. PBZ treatment was carried out weekly during 21 days. Two days after the first PBZ treatment, the seedlings were irrigated with 0 (distilled water), 75, 150 and 225 mM of NaCl solution. Salt treatment continued for 1 month. To prevent of salt accumulation, plants were leached with tap water (after every three irrigations) (Abou El- Khashab et al. 1997) and then irrigated by the same salt solution. The control plants were grown in vermiculite supplemented with Hoagland solution and sprayed with distilled water throughout the experiment. All measurements were performed 2 days after the last day of salt treatment but the weight of seeds were measured after seed maturation (140 days after sowing).

Analysis of growth

Four seedlings per replication were randomly sampled at 45 days after planting, i.e., 6th–7th leaf stage. Some growth parameters such as plant height, root length, length and area of sixth leaf, fresh and dry weight (DW) of shoot, roots and sixth leaf, and water content (WC) of total plant were measured from the collected seedlings. The plant height was measured from the base of shoot to liggule of youngest fully extended leaf on the main stem. The leaf area and the fresh weight (FW) of shoot, sixth leaf, roots and total plant were also measured. The samples were washed with distilled water and dried in an oven at 70°C for 48 h and then the DW of the samples was determined (Lindsay et al. 2004). WC of total plant was calculated using the following equation:

$$ {\text{WC (\%)}} = {\text{[(FW}} - {\text{ DW)}}/{\text{DW] }} \times {\text{ 100}} $$

Analysis of ion concentration

Oven-dried samples of the sixth leaf and roots were ground into a fine powder. For determination of Na+, K+, P and N contents, 100 mg of ground dry material of the leaf or roots was digested by adding 4 ml HClO4 at 200°C until a colorless liquid was achieved. After digestion, each sample was brought up to a 25 ml final volume with distilled-deionized water. The Na+ and K+ contents were determined with a flame photometer (Sherwood 420, Sherwood Scientific Ltd., Cambridge, UK) (Cottenie 1988; Isac and Robert 1990; Waling et al. 1989). The samples were analyzed for the N content using Kjeldahl method (Cottenie 1988; Isac and Robert 1990; Jackson 1997) and for the P content by spectrophotometeric (PG Instruments Limited T60, UV/visible spectrophotometer, London, England) method (Champan and Pratt 1961; Jackson 1997).

Statistical analysis of data

The statistical analysis was conducted using the SPSS (version 12) software package. The General Linear Models procedure was used to examine the effect of salinity and PBZ treatment on cultivars and their interactions (Klein et al. 2002), and significant difference between means were determined by one-way ANOVA based on Duncan test at α ≤ 0.05.

Results

Data indicated that salt stress significantly (P ≤ 0.05) decreased the plant height, and length and area of sixth leaf in both cultivars, particularly in Ghods cultivar (Fig. 1). At the highest level of salinity, the plant height, and sixth leaf length and area of salt-sensitive cultivar decreased by an average of 15, 12, and 33%, respectively, more than the salt-tolerant cultivar. In the both cultivars, PBZ treatment resulted in a significant reduction (P < 0.05) in the plant height, and length and area of sixth leaf (Fig. 1). The data indicated that PBZ treatment reduced the effect of salinity on plant height, length and area of sixth leaf and minimized the effect of NaCl.

Fig. 1
figure 1

Effect of salt stress (0, 75, 150 and 225 mM) and paclobutrazol treatment (0 and 30 ppm) on the plant height (A), sixth leaf length (B), sixth leaf area (C) and root length (D) in a salt-tolerant (Karchia-65) and salt-sensitive (Ghods) cultivars of wheat at 45 days after planting

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By increasing salinity, the root length decreased in both cultivars (Fig. 1). The effect of PBZ treatment on the root length was different among cultivars. PBZ treatment increased the root length in Ghods cultivar and reduced it in Karchia cultivar. The interaction of PBZ treatment and salinity moderated the negative effect of salinity on the root length. For example at 225 mM NaCl, the root length of PBZ-treated plants was significantly (P < 0.05) longer than unPBZ-treated plants.

In the both cultivars, the fresh and DW of shoot, roots and sixth leaf, and WC of total plant reduced by increasing salinity from 75 mM to 225 mM (Figs. 2 and 3). The negative effect of salinity on the fresh and DW of plants in the salt-sensitive cultivar was more than in the salt-tolerant cultivar, particularly at the highest level of salinity. PBZ application reduced the negative effect of salinity on the fresh and DW of shoot, roots and sixth leaf. In PBZ-treated plants, the fresh and DW of shoot, roots and sixth leaf improved by increasing salinity or less reduction was observed (Figs. 2 and 3). For example, the DW of roots increased in both salt-tolerant and salt-sensitive cultivars in response to PBZ treatment by 4 and 15% at 75 mM NaCl, 12 and 34% at 150 mM NaCl, and 26 and 30% at 225 mM NaCl, respectively. The effect of PBZ on controlling salinity for FW of shoot, roots and sixth leaf showed a similar tendency in the both cultivars, but its effect in the salt-sensitive cultivar was much higher (P < 0.05) than the salt-tolerant cultivar. The most positive effect of PBZ treatment on salinity tolerance was observed for the FW of roots. Salt stress significantly (P < 0.05) decreased the roots FW in both cultivars, while in presence of PBZ it showed a slight improvement by increasing salinity. PBZ-treated plants had lower WC compared with the control (Fig. 2).

Fig. 2
figure 2

Effect of salt stress (0, 75, 150 and 225 mM) and paclobutrazol treatment (0 and 30 ppm) on the fresh weight of shoot (A), roots (B) and sixth leaf (C), and water content (WC) of total plant (D) in a salt-tolerant (Karchia-65) and salt-sensitive (Ghods) cultivars of wheat at 45 days after planting

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Fig. 3
figure 3

Effect of salt stress (0, 75, 150 and 225 mM) and paclobutrazol treatment (0 and 30 ppm) on the dry weight of shoot (A), roots (B) and sixth leaf (C) in a salt-tolerant (Karchia-65) and salt-sensitive (Ghods) cultivars of wheat at 45 days after planting

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In Ghods cultivar, the stressed plants with 225 mM NaCl dried at flowering stage. Salt stress decreased the weight of seeds in both cultivars, except at 75 mM NaCl in Karchia cultivar (Fig. 4). PBZ treatment significantly (P < 0.05) increased the weight of seeds in the both cultivars (Fig. 4). PBZ treatment limited the negative effect of salinity on the weight of seeds, particularly in Ghods cultivar.

Fig. 4
figure 4

Effect of salt stress (0, 75, 150 and 225 mM) and paclobutrazol treatment (0, 30 ppm) on the weight of seeds in a salt-tolerant (Karchia-65) and salt-sensitive (Ghods) cultivars of wheat at 140 days after planting

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Salt stress increased the Na+ content of sixth leaf and roots in the both cultivars, except at 75 mM NaCl in Ghods cultivar (Fig. 5). The most significantly (P < 0.05) increased value was observed at 225 mM NaCl in Ghods cultivar. At the highest level of NaCl, the Na+ content in Karchia and Ghods cultivars was maintained by about 2- and 8-fold in the sixth leaf, and 2- and 4-fold in the roots, respectively. As expected, the roots accumulated more Na+ than the sixth leaf. Salinity significantly (P < 0.05) increased the K+ content of sixth leaf in Karchia cultivar while no significant changes was observed in the roots (Fig. 5). NaCl treatment decreased the K+ content of sixth leaf and roots in Ghods cultivar.

Fig. 5
figure 5

Effect of salt stress (0, 75, 150 and 225 mM) and paclobutrazol treatment (0 and 30 ppm) on the Na+ content of sixth leaf (A) and roots (B), and the K+ content of sixth leaf (C) and roots (D) in a salt-tolerant (Karchia-65) and salt-sensitive (Ghods) cultivars of wheat at 45 days after planting

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Data of P and N contents in the sixth leaf and roots are presented in Fig. 6. In Karchia cultivar, the P content of sixth leaf significantly increased at 225 mM NaCl, but there were no significant changes in the roots. Salt stress reduced the P content of sixth leaf and roots in Ghods cultivar. In the salt-tolerant cultivar, the N content of sixth leaf and roots significantly (P < 0.05) increased at 150 and 225 mM NaCl. In the salt-sensitive cultivar, the N content of sixth leaf in stressed plants was significantly (P < 0.05) less than in the control plants, while salinity increased it in the roots. In the control plants of both cultivars, accumulation of K+, P and N in the roots was less than the sixth leaf. In the salt-sensitive cultivar, the harmful effect of salinity on the storage of K+, P and N in the sixth leaf was significantly (P < 0.05) greater than in the roots.

Fig. 6
figure 6

Effect of salt stress (0, 75, 150 and 225 mM) and paclobutrazol treatment (0 and 30 ppm) on the P content of sixth leaf (A) and roots (B) and the N contents of sixth leaf (C) and roots (D) in a salt-tolerant (Karchia-65) and salt-sensitive (Ghods) cultivars of wheat at 45 days after planting

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PBZ application reduced the harmful effect of salinity as judged by accumulation of Na+ in the sixth leaf and roots in Ghods cultivar, and the most significant (P < 0.05) effect was observed at 225 mM NaCl (Fig. 5). At 225 mM NaCl, PBZ treatment decreased the Na+ content of sixth leaf and roots in Ghods cultivar by about 2- and 3-fold, respectively, compared with the same salinity level. Against Ghods cultivar, the interaction of PBZ treatment and salinity was not obvious on the Na+ content of sixth leaf and roots in Karchia cultivar. PBZ with or without NaCl treatment significantly (P < 0.05) increased the storage of K+ in sixth leaf of Karchia cultivar, but no significant difference was achieved in the roots (Fig. 5). PBZ treatment changed the effect of salinity on K+ and P contents in Ghods cultivar (Figs. 5 and 6). In Ghods cultivar, the K+ and P contents of PBZ-treated plants were enhanced by increasing salinity, compared with unPBZ-treated plants. At 225 mM NaCl in the salt-sensitive cultivar, PBZ treatment significantly (P < 0.05) increased the K+ content by about 50, and 25%, and P content by 77 and 20%, in the sixth leaf and roots, respectively. PBZ treatment reduced the P content of sixth leaf in Karchia cultivar while increased it slightly in the roots (Fig. 6).

Salinity with PBZ application noticeably (P < 0.05) increased the N content of sixth leaf and roots in Karchia cultivar, compared with the control (Fig. 6). PBZ with or without NaCl treatment significantly (P < 0.05) reduced the N content of sixth leaf in Ghods cultivar. In the salt-sensitive cultivar, the roots of PBZ-treated plants significantly (P < 0.05) accumulated more N content than the control. In PBZ-treated plants, the N content of roots in Ghods cultivar showed no significant changes by increasing salinity.

Discussion

Results of the present study demonstrated that PBZ treatment and salt stress significantly affected the plant growth. In saline environment where salts are present in high concentration, plant growth is affected negatively in various ways such as osmotic effect, specific ion effect and nutritional imbalance, probably all occurring simultaneously (Flowers et al. 1991). Initial growth inhibition in saline environment is induced by the decreased water potential of root medium due to high salt concentration (Munns et al. 1995). The negative effect of salt stress in Ghods cultivar was more than that of Karchia cultivar, especially at 225 mM NaCl. Pustini (2001) reported similar adverse effect of salinity on growth of salt stressed plants. Increasing NaCl concentration slowed down water uptake by plants, thereby decreasing WC, which was followed by reduction of FW in the both cultivars, particularly in the salt-sensitive cultivar. Inhibition of growth and decrease in WC induced by water stress has been universally observed even in tolerant plants (Bartls and salamini 2001; Mittler et al. 2001).

PBZ treatment influenced the measured growth parameters in the both cultivars. The effect of PBZ treatment on plant height, length and area of sixth leaf was more noticeable (P < 0.05) compared with salt stress. Reduced height is a consequence of Triazole-induced GA inhibition, exemplified by reduced internodal elongation (Fletcher et al. 2000). PBZ reduces cell number and length, so PBZ-treated plants characteristically have smaller leaf (Fletcher et al. 2000).

Measurements of root length indicated that PBZ improved the root length of Ghods cultivar in salt-stressed plants. It would be associated with larger parenchyma cells and the promotion of radial cell expansion (Fletcher et al. 2000). PBZ application reduced the negative effect of salinity on the FW of shoot in both cultivars. In Ghods cultivar, one positive effect of PBZ treatment was increasing of the roots FW by increasing salinity against un-PBZ treated plants. The leaves of PBZ-treated plants were significantly smaller than unPBZ-treated plants, therefore the fresh and DW of leaves decreased. Decreased DW of shoot correlated with decreasing of plant height and smaller leaf in PBZ-treated plants. It is hypothesized that protection of salinity in PBZ treated plants was associated with longer roots and smaller leaves for absorbing more water and loosing less water, which improve salt tolerance in salt-stressed plants. However, the WC of PBZ treated plants was less than control, it reduced slower by increasing salinity. The reduction in WC maybe associated with reduced plant size in PBZ-treated plants. Although PBZ treatment decreased the total biomass production, it improved the weight of seeds in both cultivars against stress damage. The effect of PBZ treatment on enhancing the weight of seeds in Ghods cultivar was more than Karchia cultivar.

The Na+, K+, P and N contents in investigated cultivars were strongly affected by both salinity and PBZ treatment. A secondary effect of high concentrations of Na+ and Cl− in the root medium is the suppression of uptake of essential nutrients such as K+, Ca++ and NO3 − (Perviaz et al. 2002). As expected, salinity significantly increased the Na+ content and decreased the K+ content of sixth leaf and roots in the salt-sensitive cultivar. Na+ competes with K+ for uptake through common transport systems and this happened effectively since the Na+ in saline environments is usually considerably greater than K+ (Maathuis et al. 1996; Rains and Epstein 1967). Karchia cultivar accumulated less Na+ content compared with Ghods cultivar. Salt-tolerant plants have low Na+ content in the leaf and stem, suggesting that these genotypes had a better ability to exclude harmful ions from the shoot (Lindsay et al. 2004). In Ghods cultivar, PBZ application consistently reduced the Na+ content to a level similar to the salt-tolerant cultivar. Sodium exclusion from the transpiration stream reaching the leaf is controlled at three stages: (1) by selectivity of root cells taking up cations from the soil solution, (2) by selectivity in the loading of cations into the xylem vessels in the roots, and (3) by removal of sodium from the xylem in the upper part of roots and the lower part of the shoot (Munns et al. 2002; Tester and Devenport 2003). Similar to Karchia cultivar, the K+ and P contents of PBZ-treated plants improved by increasing salinity in Ghods cultivar. In the salt sensitive cultivar, PBZ treatment reduced the changes in N content among various levels of NaCl. Tolerance mechanisms can be categorized as those that function to minimize osmotic stress or ion disequilibrium or alleviate the consequent secondary effect caused by these stresses (Bohnert et al. 1995).

Although PBZ treatment reduced the plant growth, the differences in growth among various levels of NaCl reduced in PBZ-treated plants. PBZ treatment improved the weight of seeds in both cultivars, particularly in Ghods cultivar. PBZ treatment reduced the accumulation of harmful Na+ ion in plant tissues while it increased the K+, P and N contents. It is reasonable to suggest that PBZ treatment may increase tolerance by diminishing nutritional imbalance in wheat caused by salt stress. PBZ has the potential to increase the productivity of wheat in the saline areas where high salinity limits its production. Further experiments are suggested to explore the additional pathways responsible for salt tolerance in PBZ-treated plants.