Salt stress is one of the major environmental constraints limiting affecting plant growth, development, and crop productivity  . Plant growth is compromised by salinity at all developmental stages, but sensitivity varies greatly at different stages   . This complex abiotic stress affects osmotic and ionic properties and induces a wide range of metabolic perturbations in higher plants altering many physiological and biochemical processes such as mineral nutrition, respiration rate, organic solutes/osmolyte synthesis, seed germination, enzyme activities and photosynthesis  . Nevertheless, plants have developed several adaptative mechanisms in order to survive in the presence of salt. These strategies however vary depending on plant species and even, from cultivar to cultivar  . Salt tolerance is a complex mechanism, involving tolerance to both the osmotic and ionic stresses caused by high soil salinity . The high salt concentration in plant growth environment commonly induces an increase in cellular concentrations of Na+ and Cl− and a decrease of K+ concentrations     . The regulation of Na+ and Cl– uptake is thus essential in order to avoid toxic Na+ accumulation in leaves, and to maintain a high K+/Na+ ratio which is important for the activity of K+-dependent enzymes . In many glycophyte species, such as rice, wheat, cotton and Lotus genotypes, Na+ exclusion from growing tissues (leaves or calli) has been shown to promote salt tolerance      . However, for other plants, salt-tolerance is associated with inclusion of these toxic ions; thus more tolerant types are those which accumulate more toxic ions (mainly Na+) in growing and photosynthetic tissues. In this category, we can quote sugarcane  and lens : in this case, salt-tolerance is directly linked to compartmentation of toxic ions in vacuoles in order to maintain low Na+ concentration in the cytosol compatible with enzymatic activities. Furthermore, salt tolerant genotypes maintain a high supply of K+ in the presence of an excess of Na+  . Considerable interest has been focused on Ca2+ due to its ability to induce a protective effect on plants under adverse environmental conditions. Calcium plays a vital role in salt stress tolerance since it induces antioxidant enzyme activities and reduces lipid peroxidation of cell membranes under abiotic stress  . It has also been shown to stabilize cell membrane surfaces, prevent solute leakage from the cytoplasm, maintain normal photosynthesis, regulate the plant hormone metabolism and is directly involved in transduction mechanisms  . Numerous data suggest that Na+ competes with Ca2+ for binding sites under salinity conditions and that apoplastic Ca2+ directly alleviates symptoms resulting from mineral toxicities  .
The accumulation of some organic solutes (compatible osmolytes such as proline and non-reducing sugars) also enables plants to tolerate stress effects . Proline and soluble sugars accumulation are frequently reported in plant exposed to salt or water stresses. Cytosolic accumulation of these organic solutes is indeed involved in osmotic adjustment    . Proline is also supposed to act as protective agent to enzymes, intracellular structures and cellular homeostasis balance and signaling  . The significance of proline accumulation is still a matter of debate and was reported to be a symptom of injury as reported by Lutts et al.  and Gandonou et al. . Salt stress also usually results on soluble sugars increase  . These solutes could also be involved in osmotic adjustment and tolerant varieties always accumulate more soluble sugars in leaves and growing tissue than sensitive ones    .
Contrary to several plants such as rice, corn and wheat, little is known about the physiological mechanisms involved in amaranth salt resistance. In Benin, amaranth species are mainly cultivated as leaf vegetable in the cultivable lands of the coastal areas where soil and irrigation water’s salinity constitute a major problem hampering crop production. Some studies analyzed salt effects on amaranth seed germination, plant growth and metabolism      , without however highlighting the physiological mechanisms involved in salt resistance. Data are especially crucially lacking for the species Amaranthus cruentus which is a promising plant species for saline agriculture. In a previous study, we have demonstrated that there is a variability of relative salt-stress resistance among A. cruentus cultivars at young plant stage and cv. Rouge appeared as the most salt resistant whereas cv. Locale was the most salt sensitive . In the present study, we compared NaCl effects on growth and Na+, K+, Ca2+, Mg2+, proline and soluble sugars accumulation in leaves and roots of plants of two amaranth cultivars which differ in their response to salt stress. Our goal in this study is to analyze the implication of ions and organic solutes accumulation in Amaranthus cruentus salt resistance.
2. Materials and Methods
2.1. Plant Material
Two Amaranthus cruentus cultivars were used: Locale (salt-sensitive) and Rouge (salt-resistant) according to . Species identification was performed by the team at the National Herbarium of Benin. Cultivars were obtained from the Market Gardening Crops Program of the Benin National Institute for Agricultural Research (INRAB).
2.2. Experimental Conditions
Seeds of both the cultivars were germinated in jars filled with substrate (Substrate NFU 44-551) for a 2 weeks in a greenhouse characterized by a 24/22˚C (day/night) temperature, a 16/8 hours (day/night) photoperiod, a light intensity of 120 µmole∙m−2∙s−1 provided by white fluorescent tubes (F36W/840-T8). The substrate’s pH (H2O) was 6.5 with a conductivity of 40 mS∙m−1 and a water retention capacity of 750 ml∙L−1. It contained fertitilizer (1 kg∙m−3), dry matter/raw product (50%) and organic matters/dry product (85%). The obtained young seedlings were individually transferred in pots containing the same substrate for two further weeks in a same greenhouse. Daytime humidity was set to c.a. 65%. Plants were then transferred to tanks containing a modified Hoagland solution (44) with pH 6. Stress application was carried out after one week on 5-weeks old plants. Treatments consisted in three NaCl concentrations: 0, 30 and 90 mM corresponding to an electrical conductivity of 1.91, 4.81 and 11.70 dS∙m−1, respectively. All treatments were repeated three times in a complete randomized design and each repetition consisted in a pooled sample of six plants. Eighteen plants were thus considered per treatment. Salinity stress was maintained over a period of two weeks.
2.3. Growth Determination
Plants height (aerial part) was measured before transferring to the media (H0); height was measured again after 2 weeks of treatment (H1). Relative height growth of plants (RHG) was calculated as (H1 − H0)/H0. After 15 days, the plants were harvested. Shoot and root fresh mass were determined. Samples were then transferred to an oven at 80˚C for 72 hours for dry mass determination. Data in presence of NaCl were expressed in percentage of that of the control.
2.4. Mineral Content
Ions determination was performed according to Prodjinoto et al. . Samples (c.a. 100 mg DW) were placed in flask of 10 ml and digested with nitric acid (68%) at 80˚C. After complete evaporation, residues were dissolved with nitric acid (HNO3) (68%) + HClcc (1:3, v/v). Solution was filtered using a layer of Whatman (85 mm, Grade 1). The filtrate was used to determine the cations concentration (K, Na, Mg and Ca) by flame emission using atomic absorption spectrometry (Thermo scientific S series model AAS4). The analysis was performed on 3 plants per treatment and each sample was analyzed in triplicate. Results are expressed in mg∙g−1 DW.
2.5. Extraction and Soluble Solutes Determination
Proline concentration was determined using the method of Bates et al. . Proline was extracted from organ samples (c.a. 200 mg FW) with 10 mL of 3% sulphosalicylic acid at 70˚C for 30 min. After addition of acid ninhydrin and glacial acetic acid to the extracts, the mixture was heated at 90˚C for 1 h in water bath. The reaction was then stopped by incubation for 1 h in an ice bath. The mixture was extracted with toluene and the absorbance of the toluene fraction was spectrophotometrically determined at 520 nm. Proline concentration was determined using calibration curve and expressed as μg∙proline∙g−1 FW.
Total soluble sugars were estimated by the anthrone reagent method using glucose as the standard according to  as used by Manaa et al. . Soluble sugars concentration was expressed as μg soluble sugars∙g−1 FW.
2.6. Statistical Analysis
Data are expressed as mean ± standard error with a reading of three samples per treatment. The analysis of the main effects of stress intensity and/or cultivars was based on a one-way or two-ways analysis of variance (ANOVA). Means were compared using Students, Newman and Keuls test. All statistical analyses were performed using “JMP Pro 12” soft-ware .
3.1. Plant Growth
NaCl stress significantly reduced RHG in the salt-sensitive cultivar Locale (36.6%) while no reduction was observed in the salt-resistant cultivar Rouge (Table 1). Salt effect induced a significant reduction in shoot and root fresh and dry mass in both cultivars. The average reductions due to NaCl stress were 30%, 31%, 48% and 34% for cultivar Locale and only 19%, 26%, 34% and 35% for cultivar Rouge, respectively for shoot fresh mass, shoot dry mass, root fresh mass and root dry mass (Table 1). Thus, except for root dry mass, biomass reduction by NaCl stress was more accentuated in the salt-sensitive Locale compared to the salt-resistant Rouge.
3.2. Plant Water Content
NaCl stress induced similar effect on shoot water content in both cultivars characterized by slight increase at 30 mM and no effect at 90 mM (Table 2). Thus,
Table 1. Relative Height growth (RHG), shoot fresh mass (SFM), shoot dry mass (SDM), roots fresh mass (RFM) and roots dry mass (RDM) of plants of two amaranth cultivars (Locale, salt-sensitive and Rouge, salt-tolerant) as affected by 30 and 90 mM NaCl after 2 weeks: Values are expressed as percentages (%) of means of control plants.
Table 2. Effect of different NaCl concentrations on shoot water content (%) of two cultivars of Amaranthus cruentus after two weeks stress application.
shoot water content did not change significantly under salt stress in both cultivars.
3.3. Ion Content
In the absence of stress, leaves and roots of both cultivars contained similar amounts of Na+ and K+ in leaves (Figure 1 and Figure 2). For K+ in roots and Mg2+ in leaves and in roots, the resistant cultivar Rouge contained more ion than the salt sensitive cultivar Locale in the absence of stress while the sensitive cultivar Locale contained more Ca2+ than the resistant Rouge either in leaves or in roots (Figures 2-4). A dose dependent increase in Na+ (Figure 1) concentration was recorded in leaves and in roots while a concomitant decrease was observed for K+, Ca2+ and Mg2+ concentrations in both cultivars (except K+ in leaves at 30 mM for the resistant cultivar) (Figures 2-4). A two-way ANOVA showed a significant effect of NaCl (p < 0.001) on Na+ concentrations in both cultivars in leaves and in roots; statistical analysis revealed also no significant difference between the two cultivars in leaves and roots Na+ concentrations and no interaction between NaCl concentrations and cultivars (Table 3). However, data revealed that the increase of Na+ concentration was more accentuated in the salt resistant
Figure 1. Effect of NaCl salt stress on sodium ion (Na+) content in two cultivars of amaranth after 2 weeks: (a) leaves; (b) Roots.
Figure 2. Effect of NaCl salt stress on potassium ion (K+) content in two cultivars of amaranth after 2 weeks: (a) leaves; (b) Roots.
Figure 3. Effect of NaCl salt stress on calcium ion (Ca2+) content in two cultivars of amaranth after 2 weeks: (a) leaves; (b) Roots.
Figure 4. Effect of NaCl salt stress on magnesium content in two cultivars of amaranth after 2 weeks: (a) leaves; (b) Roots.
Table 3. Results of 2-ways variance analysis for ion content, proline and soluble sugars accumulation of leaves and roots of plants from two cultivars of amaranth; F-ratios are given for the main effects of the following levels of classification: stress intensity (i.e. NaCl concentration of stressing media), cultivars and interaction between these levels of classification.
ns: not significant; *: significant at p = 0.05; **: significant at p = 0.01; ***: significant at p = 0.001.
cv. Rouge (respectively 510% and 3469% of that of the control) than in the salt sensitive cv. Locale (respectively 278% and 2921% at 30 and 90 mM NaCl) in leaves. In roots, the same tendency was observed with an increase corresponding to 4161% and 5312% of that of the control for the salt-resistant cv. Rouge and to 1072% and 1539% for the salt-sensitive cv. Locale.
A two-ways ANOVA showed a significant effect of NaCl (p < 0.001) only for root K+ concentrations; no significant difference was observed between the two cultivars in leaves and roots K+ concentrations and no interaction between NaCl concentrations and cultivars (Table 3). However, data revealed a slight increase (4%) of K+ concentration in leaves of the salt resistant cv. Rouge at 30 mM NaCl followed by a slight decrease (5.5%) at 90 mM NaCl while a continuous decrease (6% and 9% respectively with 30 and 90 mM NaCl) was observed for the salt sensitive cv. Locale (Figure 2). In roots, a continuous decrease was observed in both cultivars and such a decrease was more accentuated in roots of the salt-resistant cv. Rouge than in salt-sensitive cv. Locale. Thus, the reduction of K+ content by salt stress was, from a relative point of view, less accentuated in leaves of the salt-resistant cv. Rouge than the salt-sensitive cv. Locale whereas an opposite tendency was observed in roots. A two-ways ANOVA also showed a significant reduction of Ca2+ concentrations in both cultivars in leaves (p < 0.001) and in roots (p < 0.01) under salt stress. However, the one-way ANOVA revealed that the reduction was significant (p < 0.05) only in leaves and roots of the salt-resistant cv. Rouge (Figure 3). Moreover, the salt sensitive cv. Locale presented the highest Ca2+ concentrations in both leaves and roots at all NaCl concentrations used.
A two-ways ANOVA showed a significant reduction of Mg2+ concentrations in leaves of both cultivars (p < 0.05) and a significant difference was recorded between cultivars (p < 0.01) for leaves Mg2+ concentrations (Table 3). The reduction of leaf Mg2+ concentration under salt-stress was more accentuated in the salt sensitive cv. Locale (Figure 4) and only a slight increase were observed at 30 mM NaCl for the salt-resistant cultivar. For roots Mg2+ concentrations, a significant interaction (p < 0.05) was observed between stress and cultivars. The one way ANOVA revealed that the reduction of roots Mg2+ concentration under salt-stress was significant only in the salt sensitive cv. Locale. A slight increase was observed at 90 mM NaCl for the salt-resistant cultivar (Figure 4). However, the salt sensitive cv. Locale presented the highest Mg2+ concentrations in both leaves and roots at all NaCl concentrations used (except 90 mM in roots). Thus, the effect of stress on Mg2+ content was more accentuated in the salt-sensitive cv. Locale than in the salt-resistant cv. Rouge either in leaves or in roots but the salt resistant cultivar Rouge accumulated less Mg2+ in both leaves and roots.
3.4. Free Proline and Soluble Sugars Accumulation
Figure 5. Effect of NaCl salt stress on proline content in two cultivars of amaranth after 2 weeks: (a) leaves; (b) Roots.
Figure 6. Effect of NaCl salt stress on soluble sugars content in two cultivars of amaranth after 2 weeks: (a) leaves; (b) Roots.
absence of NaCl. In the presence of NaCl, free proline increased in leaves (7.75% and 345% respectively at 30 and 90 mM NaCl) and roots (103.6% and 255% respectively at 30 and 90 mM NaCl) of the salt-sensitive cv. Locale. A two-way ANOVA revealed significant (p < 0.01) effect of NaCl and a significant (p < 0.05) difference between cultivars for both leaves and roots proline content under salt stress and no interaction (leaves) and a significant interaction (p < 0.01) between NaCl concentrations and cultivars (Table 3). These results indicated that the increase of free proline content under salt stress was more accentuated in both leaves and roots of the salt-sensitive cv. Locale than the salt resistant Rouge. However, the salt resistant cv. Rouge presented the highest proline concentrations in both leaves and roots at the lowest NaCl concentrations used (0 and 30 mM NaCl) whereas an opposite tendency was observed at the highest NaCl concentration used (90 mM).
In the presence of NaCl, soluble sugars markedly increased in leaves of both cultivars (Figure 6). A two-way ANOVA revealed that NaCl effect on leaves soluble sugars content was significant (p < 0.001) in both cultivars with a significant difference (p < 0.01) between them and no interaction between NaCl concentrations and cultivars. The increase of leaf soluble sugars content under salt stress was more accentuated in the salt-resistant cultivar than the salt sensitive one. For roots soluble sugars content, a two-way ANOVA revealed a significant (p < 0.001) effect of salt-stress, a significant difference (p < 0.05) between cultivars and a significant interaction (p < 0.05) between salt stress and cultivars (Table 3). The one-way ANOVA for each cultivar revealed that the effect of NaCl stress on roots soluble sugars content was significant (p < 0.001) at all NaCl concentrations used in the salt-sensitive cv. Locale whereas it was non-significant in the salt resistant cv. Rouge whatever the NaCl dose (Figure 6). Moreover, the salt resistant cultivar Rouge presented the highest soluble sugars concentration in both leaves and roots at all NaCl concentrations used (except in roots at 90 mM NaCl).
Our results revealed that plant growth reduction due to NaCl stress was more accentuated in the salt-sensitive Locale compared to the salt-resistant Rouge confirming the salt-resistance status of both cultivars as previously reported . Salinity classically induced cell dehydration at low water potential, nutritional imbalance caused by the interference of saline ions with essential nutrients in both uptake and translocation processes and toxicity due to the high accumulation of Na+ and Cl− in the cytoplasm. No changes in plant water content were observed in our cultivars indicating that water content parameter is not the main aspect of salt stress effect in these cultivars, corroborating our previous finding in the cultivar Locale  .
Ion homeostasis and compartmentalization in addition to ion transport and uptake are some of the principle physiological mechanisms developped by plant in order to survive in soils with high salt concentration . Thus, Maintaining ion homeostasis can be particularly challenging for plants under saline conditions, as the accumulation of toxic ions (i.e. Na+) can perturb the plant’s ability to control accumulation of other ions. In most species, Na+ appears to accumulate to toxic levels before Cl− does  and Cl– is considered less toxic than Na+ . Thus, we focus here on Na+, because reducing Na+ in the shoot, while maintaining K+ homeostasis, is a key component of salinity tolerance in many crops . Plants of the two cultivars accumulated high amounts of Na+ in leaves when exposed to NaCl. Our results revealed that salt-resistant Rouge accumulated more Na+ than the salt-sensitive Locale in leaves and roots. These results indicated that Na+ ions toxicity should play a key role in NaCl adverse effects on amaranth plants growth. Thus, the detrimental effects of Na+ on growth are more accentuated in aerial part (RHG and SFM, mainly) of the sensitive Locale compared to cv. Rouge plants. It may be hypothesized that accumulation of these ions in leaves apparently did not cause much injury to the aerial part in resistant cultivar. Leaf Na+ exclusion has been reported as a mechanism for salt tolerance in numerous species . Na+ exclusion from leaves is associated with salt tolerance in several crops such as rice, durum wheat, bread wheat, barley, sugarcane, pearl millet    and references therein]. However, the salt resistance of the amaranth cultivar Rouge was not associated to Na+ exclusion as reported in some cultivars of other plant species including maize  and sugarcane . The critical point of plant salt tolerance was to keep Na+ and Cl– concentrations in the cytoplasm below toxic levels  by partitioning these ions within cells so that concentrations in the cytoplasm are kept low . Thus, it is logical to infer that much of the Na+ accumulated by the salt-resistant cultivar Rouge are sequestrated in the vacuoles or extruded to the apoplasm  causing very little or no interference with the cellular metabolism necessary for sustained growth. Salt-resistance cv. Rouge appeared as a toxic ions includer since its accumulated more Na+ in leaves (and roots) in presence of NaCl than the salt sensitive cultivar.
In the presence of NaCl, the salt-resistant Rouge maintained high amounts of K+ in leaves. In plants, higher K+ was considered as an important indicator of salt resistance  as potassium ions are known to be a major component of osmotic adjustment during stress . K+ concentrations in plants usually decrease with NaCl treatment . In the presence of NaCl, K+ concentration decreased significantly only in roots although the decrease was more accentuated in leaves of the salt-sensitive cultivar Locale than the salt-resistant cultivar Rouge. It is well known that Na+ and K+ cations compete for uptake by membrane transporters  and K+ deficiency relates to plants productivity as it is involved in important physiological processes, such as osmotic adjustment during stress  and stomatal opening . Thus, the salt resistance of the cultivar Rouge could partly be due to the internal maintenance of high concentrations of K+ in leaves in the presence of high amount of Na+ in the medium.
Several reports suggested that apoplastic Ca2+ directly alleviates symptoms produced by mineral toxicities under salt stress. In this study, salt stress induced a significant reduction in both leaves and roots Ca2+ concentration in both cultivars but the reduction was more accentuated in both leaves and roots of the salt-resistant cv. Rouge than the salt-sensitive cv. Locale and the salt-resistant cultivar accumulated less Ca2+ in both leaves and roots than the salt-sensitive one. This result indicated that Ca2+ uptake and transport were not implicated in the salt-resistance of the cultivar Rouge or that Ca2+ content was sufficient to provide adequate protection to salinity in this cultivar. It stated that high levels of Na+ inhibits Mg2+ in leaves  . In this study, the reduction of Mg2+ content under salt stress was more accentuated in the salt-sensitive cultivar Locale than in the salt-resistant cv. Rouge either in leaves or in roots. Thus, in Amaranthus cruentus cultivars, Mg2+ accumulation appears as an indicator of salt resistance.
Biosynthesis of osmoprotectants and compatible solutes are among the principle physiological and biochemical mechanisms developped by plants in order to survive in soils with high salt concentration . Proline and soluble sugars are the main members of these osmoprotectants and compatible solutes. Proline is thought to be involved in osmotic adjustment but it may also directly act to protect cellular structures and enzymes or scavenge reactive oxygen species . In african rice species O. glaberrima cultivars, Prodjinoto et al.  suggested that proline does not assume key functions in salinity resistance or that the signaling pathway leading to proline over-synthesis is still not triggered in salt-resistant which significantly accumulated lower proline concentrations than salt-sensitive one. A similar situation was reported in O. sativa where salt-resistant cultivars accumulated lower proline concentrations than salt-sensitive ones  as well as in sugarcane  . We demonstrated that the increase of free proline content under salt stress was more accentuated in leaves and roots of the salt-sensitive cultivar Locale than the salt resistant Rouge. Thus, in Amaranthus cruentus cultivars, proline should be regarded not as an indicator of salt resistance but as a typical “stress response” solute as reported in tomato .
Sugars are also considered to play a major role in osmoregulation under abiotic stress conditions . According to , total soluble carbohydrates are important solutes that accumulate in cytosol under salt stress and may thus contribute to plant survival. Our results revealed that NaCl induced an increase in soluble sugars content both in leaves and in roots of both cultivars. Resistant cv. Rouge accumulated more soluble sugars than the sensitive Locale in leaves; this tendency is reversed in the roots. Salt stress effects result mainly in an increase of soluble sugars content in both leaves and roots in many plants and the most tolerant cultivars frequently accumulated more soluble sugars as reported for sugarcane   . According to , among the organic osmotica, sugars contribute by more than 50% to the total osmotic potential in glycophytes exposed to salt stress. The fact that the most resistant cultivar accumulates more soluble sugars under salt stress especially in leaves seems to indicate that soluble sugars play a role of an osmo-protector in salt resistance of Amaranthus cruentus.
The present study revealed that Na+ toxicity is implied in salt effect in Amaranthus cruentus plants and that cultivar Rouge salt-resistance is due to a tolerance mechanism. This study showed that K+ and Mg2+ play an important role in Amaranthus cruentus plant salt resistance and that organic solutes such as soluble sugars would be directly involved in salt resistance in this species. Proline is not directly involved in salt resistance in Amaranthus cruentus plants.
The authors thank Mr. Armel Mensah from Market Gardening Crops Program of the Benin National Institute for Agricultural Research (INRAB) for seeds providing and Prof. Hounnankpon Yédomonhan from the National Herbarium of Benin for Amaranth species identification. This work was supported by the Programme “Formation des Formateurs” of the University of Abomey-Calavi, Republic of Benin.
RHG Relative Height Growth
SFM Shoot Fresh Mass
SDM Shoot Dry Mass
RFM Root Fresh Mass
RDM Root Dry Mass
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