Compared salt tolerance of five local wheat (Triticum aestivum L.) cultivars of Albania based on morphology, pigment synthesis and glutathione content

One hundred seeds from each of five bread wheat (T. aestivum L.) cultivars (Suba, Nogal, Dajti, Toborzo, Viktoria) donated from the Public Directory of Agriculture of Vlora, Albania, were kept at 4ᵒC for 24hrs, and later in running water for 30 min following (28).

After, seeds (25 seeds / plate) were sown in Petri dishes containing filter paper soaked in distilled water, and kept in vegetative room under the photoperiod 16/8 day-night at 25ᵒC for four days to germinate.

Thereafter, plantlets were transferred to cultures with modified Hoagland solution (29). Four different solutions containing respectively 0-50mM-100mM-200mM NaCl were used to develop plantlets during three consecutive weeks. Altogether, 4 experiments (controls plus three treatments) with four replications each, for each of the five cultivars were conducted in the growth room under a photoperiod 16/8 day-night, at 25ᵒC.

To measure the length of leaves, shoots and roots were chosen plantlets (3 paralels/each culture) 3 weeks after the application of the saline stress.

Photosynthetic pigments (chla, chlb, carotenods and xantophylls) were extracted from leaves of plantlets 2 and 3 weeks after saline treatment using a non-destructive method, by mixing dimethyl sulfoxide solution with plants tissues following the standard DMSO protocol (30) adopted by (31). For each cultivar were used 3 replicates x 4 categories of cultures (1 control and three treatments). Absorbance of the pigments extracts was measured at 470nm, 645nm and 663nm, and quantities were calculated following Arnon’s equations (32).

Plantlets aged two and three weeks after treatment with salinity were used to extract total thiols following (33) and GSH content was evaluated following Ellman’s test (34). The so-called Ellman’s reagent enters the reaction with free sulfhydryl groups to form mixed disulfides and 2-nitro-5-thiobenzoic acid. The last one has a yellowish color and its absorbance was measured at 412 nm in spectrophotometer. For each cultivar were used 3 replicates x 4 categories Data of cultures (1 control and three treatments).

Statistical analysis of biometric parameters, pigments and thiol concentrations were carried out using the IBM SPSS Statistics 20 program. It was used to verify the impact of different saline concentrations on biometric parameters, pigments, and a thiols for each cultivar, as well as to compare GSH content among cultivars, and the impact of the time of exposure to treatment.

Three biometric parameters (length of leaf, shoot and root) were measured three weeks after treatment with saline stress. Leafs length displayed a progressive reduction, in negative correlation to the increase in salt concentration, for all cultivars except Dajti, whose leaves kept the same length under the saline concentrations 100-200mM (Table 1, Fig 1).

Figure 1

The biometric parameter length of roots displayed a cultivar specific trend. All cultivars had slower root growth at samples treated with 50mM NaCl compared to controls; Suba had an almost stabilized root growth under all treatments; Dajti had a progressive reduction of root length with the increase on salt concentration; Toborzo and Nogal recuperated root growth from treatment 100mM to 200mM; Viktoria displayed the biggest reduction in root length (control plants compared to those treated with the highest salt concentration) (Table 1, Fig. 2).

Figure 2

The highest shoot length was measured at control plants for the five cultivars, and this biometric parameter followed a cultivar specific trend. Thus, Suba, Nogal and Dajti displayed (progressive reduction of shoot length with the increase of salt concentration; Viktoria had a stabilized shoot length independently of treatment with salinity; Toborzo had higher shoot length under treatment 200mM compared to 100mM (Fig. 3).

Figure 3

Results on photosynthetic pigments Chla, Chlb, carotenoids and xantophylls extracted and measured at wheat plant material under saline treatment, two and three weeks after the application are presented at Table 2 and Figure 4, 5, 6, 7.

Figure 4

Figure 5

Figure 6

Figure 7

The pigment content for cultivar Suba displayed increase in Chla in correlation with the time of exposure to salinity; increase under treatment with 50-100mM NaCl, and lowering under treatment with 200mM NaCl. The quantity of chla versus chlb varies from 2,6 – 4; Carotenoids and xantophylls increased by the time of exposure to treatment.

At plants after the 2^nd week of treatment the chla and chlb increased in positive correlation with the concentration of salinity; after the 3^rd week chla is decreased in negative correlation with salinity, while the chlb is decreased in second treatment (100mM NaCl), and kept almost the same concentration during the first and third treatment (50-200mM).

Chla and Chlb increased under first treatment (50mM) and decreased under the effect of 100-200mM NaCl in both 2^nd and 3^rd week. It is clear that the Chla/Chlb remain more stable during the 2^nd week after treatment; increase during 50-100mM treatments, and decrease under the highest saline concentration after the 3^nd week. It seems that the possible conversion of Chlb to Chla, and the reduction of Chlb synthesis is more active during the prolonged effect of salinity. Carotenoids and xantophylls content reach highest concentration at cultivar Viktoria under 200mM NaCl / 2^nd week and at 50mM salinity / 3^rd week.

From Figures 4, 5 can be depicted that Chla and Chlb quantities decrease from 2^nd to 3^rd week of treatment with salinity; A sharp decrease was evidenced at plants with 50mM NaCl treatment, followed by the gradual increase under the rest of salinity conditions.

Ratios Chla/Chlb reach maximum (for cultivar Nogal) values at plants treated with 50mM NaCl, decrease for the rest of treatments, while values remain higher for the 3^rd week (Fig. 8). It seems that, either the synthesis of Chla increases, or conversion of Chlb is favored under stress conditions.

Figure 8

Chla content decreases from 2^nd to 3^rd week of treatment; Chlb on the opposite increases with time of exposure; Both Chla and Chlb reach highest values at treatment with 50mM NaCl.

Ratio chla/chlb decreased slightly from the 2^nd to 3^rd week; Peak values were measured at plants under treatment with 100mM NaCl.

Results show a sharp decrease of carotenoids and xantophylls from the 2^nd to 3^rd week of treatment; the lowest values are reached for plants under 50mM NaCl treatment (Fig. 7).

Their concentration after the 2^nd week of treatment reaches highest values at 50mM NaCl and remains the same as controls for the rest of salinities applied; Three weeks after treatment concentration decreases for plants grown in 50-100mM and grows to the values of control plants at 200mM NaCl.

Table 3 describes results on GSH content at five cultivars under treatment with modified Hoagland solutions.

Soil salinity is a major constraint to food production because it limits crop yield, even though most halophytes and glycophytes tolerate salinity (24). Previous reports have summarized research on plant ion homeostasis in saline environments trying to integrate current understanding of salt stress sensing, which leads to the activation of the ROS pathway and the regulation of ion transport systems that facilitate homeostasis (8, 24, 26, 35).

Fundamentally, plants cope by either avoiding or tolerating salt stress (24). According to (4) the cultivation of tolerant genotypes is recognized as the most effective way to overcome limitations in wheat production in salt-affected areas. Albania is a Mediterranean country, which faces environmental stresses such as high temperatures, drought, and salinity in different regions.

In this study, five bread wheat local cultivars of Vlora were analyzed to compare their salt-related tolerance during seedling in Hoagland solutions, by investigating biometric parameters, pigment synthesis and glutathione metabolism under the effect of 50mM-100mM-200mM NaCl. Table 4 summarizes the effect of salinity concentration in root, shoot and leaf development three weeks after growth in modified Hoagland solutions.

From Table 4 can easily be depicted that cultivar Nogal experiences growth reduction for the three parameters measured; Roots development is impaired at 3/5 cultivars; Shoots development is impaired at 4/5 cultivars; Leaf development is reduced at 4/5 cultivars. As previously reported (36) the chemical potential of the saline solution initially establishes a water potential imbalance between the apoplast and symplast that leads to turgor decrease, which if severe enough can cause growth reduction. Our results prove that the effect of salinity is displayed not only in the areas exposed to salinity (roots). Following (8), plant operates as an integrated system in which metabolic stress-induced signals spread in the plant and change the metabolism even in areas in which the stress conditions are not present. Furthermore, different parts of plant root system may behave as physiologically autonomous units with different responses to environmental signals and preserving their own capability to supply shoots with water, nutrients or assimilates (8). In accordance to (10) shoot growth is more sensitive to salt stress than the root growth for the five investigated cultivars. In conclusion, the increase in salinity concentration from 50-200mM has affected growth parameters for the five cultivars; cv Nogal is the most sensitive; cv Dajti is the only which continues an unimpaired leaf development. Suba and Toborzo have a stable root development.

A summary of the results on the impact of saline stress on photosynthetic pigments for five investigated is presented at Table 5. For 3 out of 5 cultivars total chlorophylls increased; for 1 out of 5 cultivars Tot Chl decreased, and 1 out of 5 cultivars was not impacted by the exposure to salinity. According to (9), the increase in Chl content happens in salt-tolerant species, and following our results cultivars Dajti, Suba and Toborzo could be classified as such. The decrease of the ratio chla/chlb can be explained by the possible conversion of Chlb into Chla (for Dajti and Toborzo), while in the case of Suba the variation of the ratio could be explained by the degradation of Chla. Nogal seems to be the most impacted cultivar (tot chl and carotenoids synthesis decrease) meaning the most sensitive to salinity; Cultivar Viktoria displayed unchanged total Chl, and carotenoids+xantophylls meaning a stable tot chl content, which might be explained by the the ratio of Chla / Chlb (Fig. 8).

Carotenoids and xantophylls in 4 out 5 cultivars remain in constant concentrations; Nogal is the only who experiences reduction. As previously reported, these pigments are necessary for photoprotection of photosynthesis and they play an important role as precursors in signaling during the plant development under abiotic/biotic stress (9). In conclusion, the reduction of carotenoids, Chla and Chlb for cultivar Nogal, speak for an impaired pigment synthesis and lead us to classify it as salt-sensitive. The rest of cultivars could be classified as salt-tolerant cultivars because they kept an almost constant carotenoids+xantophylls content, and increased total Chl. The cultivar specific reductions of Chla or Chlb (Table 4) have been previously reported in Triticum aestivum by (9, 14). Following (14) the salt-induced alterations of leaf Chl content could be due to impaired biosynthesis or accelerated pigment degradation, Chlb may be converted into Chla, thus resulting in the increased content of Chla.

According to (26) known functions of glutathione include roles in biosynthetic pathways, detoxification, antioxidant biochemistry and redox homeostasis. Based on (27, 38) most important factors affecting synthesis of GSH are γ-ECS activity and cysteine availability. So far, the induction of the transcripts of GSH1 and GSH2 genes is reported to be caused by a few substances such as jasmonic acid, heavy metals, some special light conditions, draught, and certain pathogens. High salinity decreases the capacity of roots to extract water from soil, and high concentrations of salts within the plant itself can be toxic, resulting in nutritional imbalance and oxidative stress (7, 10).

Five cultivars under study have a decreased amount of GSH from 2^nd to 3^rd week after treatment with modified salinity; Cultivar Suba experiences progressive reduction with the increase of NaCl concentration applied; Toborzo has higher GSH content with increase of salt cc; Viktoria has lower GSH content compared to control, but almost equal cc among treatments; Nogal and Dajti reach peak GSH content at plants treated with 50mM NaCl.

As previously reported (26), mechanisms appear to have evolved to stabilize cytosolic glutathione redox potential during increased ROS production, and these include increases in total glutathione and accumulation of GSSG in compartments that may be less sensitive to redox perturbation. Based on our results, the quantity of GSH in response to different levels of salinity is cultivar specific, but the time of exposure to salinity is in negative correlation to GSH content for all investigated cultivars. As already known, glutathione accumulation, turnover and transport involve a number of enzymatic pathways many of which not elucidated, however it is well known that at cereals GSH1 and GSH2 genes regulate its biosynthesis in plastids (γ-ECS synthesis) and cytosol (26, 37). Either of the modes of regulation of glutathione involve γ-ECS, which seems to contribute for the glutathione homeostasis via a feedback inhibition mechanism. Based on (38), local wheat cultivars, grown in the absence of biotic or abiotic conditions, display typical tissue and cultivar specific accumulation of GSH.

In conclusion, based on biometric parameters, pigment synthesis and GSH content: wheat cultivar Nogal is salt-sensitive (growth and pigments reduced), cultivar Viktoria medium-tolerant (growth partially impaired, pigments constant), Toborzo and Suba medium-tolerant (growth partially impaired, pigments increased), Dajti salt-tolerant (growth partially impaired / leaves developed, pigments increased). Mechanism used to stabilize GSH content is cultivar specific.