Using Xyloglucan Oligosaccharides as Biostimulant to Enhance Tobacco Tolerance to Salt Stress- Juniper Publishers

Journal of Agriculture Research- Juniper Publishers

Abstract

Xyloglucan oligosaccharides (XGOs) derived from the hydrolysis of plant cell wall xyloglucan, are a new class of naturally occurring biostimulants that exert a positive effect on plant growth and morphology and can enhance plant’s tolerance to stress. Here, we aimed to determine the influence of exogenous Tamarindus indica L. cell wall-derived XGOs on Nicotiana tabacum’s tolerance to salt stress by examining the plant’s morphology, physiological, and metabolic changes after XGO application. N. tabacum plants were grown in solid media for two months under salt stress with 100mM of sodium chloride (NaCl) ± 0.1μM XGO. Germination percentage (GP), number of leaves (NL), foliar area (FA), primary root length (PRL), and density of lateral roots (DLR) were measured. Also, 21-old-day N. tabacum plants were treated with a salt shock (100mM NaCl) ± 0.1μM XGOs. Proline, total chlorophyll, and total carbonyl contents in addition to lipid peroxidation degree and activities of four enzymes related to oxidative stress were quantified. Results showed that under saline conditions, XGOs caused a significant increase in NL and PRL, promoted lateral root formation, produced an increase in proline and total Chl contents, while reducing protein oxidation and lipid peroxidation. Although they modulated the activity of the enzymes analyzed, they were not statistically different from the salt control. XGOs may act as metabolic inducers that trigger the physiological responses for counteracting the negative effects of oxidative stress under saline conditions.

Keywords: Antioxidant system; Biostimulants; Nicotiana tabacum; Salt stress; Xyloglucan oligosaccharides

Abbreviations: CAT: Catalase; Chl: Chlorophyll; DLR: Density of Lateral Roots; FA: Foliar Area; GP: Germination Percentage; GPX: Peroxidase; GR: Glutathione Reductase; MS: Murashige and Skoog; NaCl: Sodium Chloride; NL: Number of Leaves; PCA: Principal Component Analysis; PRL: Primary Root Length; RL: Lateral Roots; ROS: Reactive Oxygen Species; SOD: Superoxide Dismutase; XGOs: Xyloglucan Oligosaccharides

Introduction

Modern agriculture faces many challenges in order to meet the growing demand for worldwide food. The world’s population is growing at an accelerated rate. By the end of 2050 it is expected to reach 9.8 billion people and 11.2 billion in 2100 according to the “World Population Prospects: The 2017 Revision”, published by the United Nations Department of Economic and Social Affairs. However, food productivity and availability are decreasing as a result of the effects of several biotic and abiotic factors. Therefore, several actions are being taken to reduce these losses and to cope with the growing food need for the world’s population.

Soil salinity is a worldwide phenomenon that occurs under almost all climatic conditions and is a major impediment to achieving increased crop yields. Using the FAO/UNESCO soil map of the world (1970–1980), FAO estimated that 19.5% of irrigated land were salt-affected soils, and of the almost 1.5 billion ha of dryland agriculture, 32 million (2.1%) suffer from salinity problems [1]. Salt-affected soils are characterized by abundant quantities of neutral soluble salts that adversely affect plant uptake of nutrients in the soil and their growth [2]. Under salt stress, plants are also under other types of stresses, which have deleterious effects on them such as water stress, ionic toxicity, and nutritional deficiencies [2]. Altogether, these conditions confer oxidative stress and metabolic imbalance to plants [3]. Consequently, plants exposed to high saline conditions shown growth inhibition or retardation.

The morphology of plants exposed to salinity can be affected by soil salt concentrations, type of plant species, age, and plant stages (vegetative or flowering), and/or the type of salt present [4,5]. For example, there is a decrease in plant lengths, leaf (foliar) areas, leaf numbers and root systems under high concentrations of NaCl [4]. Also, many studies confirm the inhibitory effects of salinity on photosynthesis by changing chlorophyll content thus affecting Chl components and damaging the photosynthetic apparatus [5].

In addition, plants exposed to high NaCl concentrations (such as100-200mM) show rapid overproduction of reactive oxygen species, which have detrimental effects on the plants’ cells. ROS causes membrane lipid component peroxidation and oxidation of cellular components such as proteins and nucleic acids, which finally lead to programmed cell death [6,7]. ROS-initiated damage is reduced and repaired by a complex antioxidant system, which combines enzymatic and non-enzymatic components. It consists of low molecular weight antioxidant metabolites, including ascorbic acid, carotenoids, glutathione, α-tocopherol and enzymes such as catalase, peroxidase, superoxide dismutase, glutathione reductase and others. The degree of cellular damage will depend on the balance between ROS production and elimination by the antioxidant scavenging system [8].

Plants also accumulate compatible solutes in response to salt stress, which provides protection to them by participating in ROS detoxification and cellular osmotic regulation in addition to contributing to enzyme/protein stabilization and membrane integrity protection [6]. Among them, proline is one of the most important ones due to its multiple roles as part of the plant’s response to various types of stresses. It functions as an osmolyte for osmotic adjustment, buffering cellular redox potential under stress conditions, maintaining protein integrity, enhancing different enzymes activities, and free radical scavenging [9,10]. Its accumulation in leaves under salt stress has been correlated with stress tolerance in many plant species, allowing them to survive under this type of stress [6].

Many efforts have been done to overcome the problems associated with high soil salinity and salt stress in plants. However, the use of traditional physical and chemical methods for environmental restoration of salt contaminated soils demand significant investment of technological and economic resources [11]. In addition to these traditional approaches, different biostimulant classes have been used to increase crop performance under salt stress and to mitigate stress-induced limitations [12-14]. A plant biostimulant is any substance or microorganism that is applied to plants with the aim of enhancing nutrition efficiency, abiotic stress tolerance and/or crop quality traits regardless of its nutrients content. By extension, they also designate commercial products containing mixtures of such substances and/or microorganisms [15]. Plant biostimulants based on natural materials have received considerable attention by both the scientific community and commercial enterprises. According to Stratistics Market Research Consulting (MRC), the Global Biostimulants Market is accounted for $1.50 billion in 2016 and is expected to grow gradually to reach $3.79 billion by 2023 due to growing importance for organic products in agricultural industries [16]. However, understanding the mechanisms by which biostimulants act is critical to their widespread use for helping plants cope in saline-affected soils.

XGOs, derived from the breakdown of xyloglucans in plant cell walls, are emerging as a new class of naturally occurring biostimulants as a result of their positive effects on plant growth and morphology [17-19]. Plant-derived XGOs are also used as biotic pesticides and seed coating agents to maintain plant freshness in addition to capsule materials for synthetic seeds [20]. Xyloglucan is the quantitatively predominant hemicellulosic polysaccharide in the primary walls, which consists of ~20% (w/w) dicot and ~5% monocot primary cell walls [21]. Its backbone is composed of a β-(1,4)-D-glucan backbone that is quasi-regularly substituted with α-D-xylosyl residues linked to glucose through the O-6 position. In many species, the backbone has a regular pattern of three substituted glucose units followed by an unsubstituted glucose residue [22]. As a variety of complex structures can be formed, a code letter for each glucosyl residue has been defined to allow for the unambiguous naming of xyloglucan oligosaccharides. For example, XGOs can be classified as the XXXG-type of the XXGG-type, in which a capital G represents a unbranched Glcp residue and a capital F represents a Glcp residue that is substituted with a fucose- containing trisaccharide [23]. Soluble XGOs can be obtained from tamarind (Tamarindus indica L.) seeds after partial digestion with cellulase. A fraction of these XGOs have been shown to have physiologically active functions in plants and oligosaccharides, also known as oligosaccharins [19,24]. Their biological properties in plants depends on the fragmented structures and their concentrations, which need to be extremely low to get a variety of effects (10–9 - 10–8M) [17-19]. Few experimental data are available concerning the use of the XGOs as plant biostimulants for mitigating the damage imposed by salt stress conditions in plants [25]. Also, each new formulation requires a new biological evaluation to ensure that the effects are beneficial, consistent, and predictable.

For all of the above, the objective of this study was to determine the biostimulating effects of application of exogenous XGO derived from T. indica L. cell walls on N. tabacum seedlings grown under saline stress conditions with special attention to their influence on plant morphology, necessary physiological and metabolic changes to overcome stress, ROS detoxification, and antioxidant capacities.

Materials and Methods

Oligosaccharin composition and concentration used

The XGO fraction used in this work was the same formulation previously reported and tested on plants [18,26]. Briefly, XGO was extracted and purified from tamarind (T. indica L.) seeds. The predominant composition of the XGO extracts consisted of XLLG and XXLG/XLXG with lower proportions of XXXG, XXGG, and XXG oligosaccharides as classified by Fry et al. [23]. Mass spectra obtained by matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) spectrometry [27] are shown in Supplementary Table 1&1A. Relative proportions of xyloglucan oligosaccharides obtained by MALDI and high-performance anion-exchange chromatography with pulsed amperometric detection analysis were similar (data not show). The isolated XGO fraction showed no cellulase activity, and protein could not be detected. The uniformity of the XGO mixture was confirmed by gel filtration analysis through a BioGel P2 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). XGOs were used at a final concentration of 0.1μM which was selected based on previous experiments as an optimal concentration for stimulating root and leaf development in N. tabacum without causing changes in plants’ chromosome number [17,28].

Plant material and general growth conditions

Botanical seeds of N. tabacum Linn. were used as the plant model. Seeds were kindly provided by Dr. Alexis Acosta Maspons from the Institute of Biotechnology of National Autonomous University of Mexico (UNAM) (Cuernavaca, Morelos, Mexico). All seeds were harvested at the same time, kept at 4 °C in the dark, and grown under the same controlled conditions. Seeds were surface- sterilized and grown on MS [29] solid medium in a growth chamber (DAIHAN Scientific, model WISD, Korea) at 23 °C in longday conditions (16h light/8h dark) and 50% relative humidity. Sowing and the way in which each treatment was applied was according to the chosen evaluation (plant and root morphology measurements and biochemical analysis) and is explained in detail for each one in this section. Each experiment was repeated to generate three biological replicates.

Germination percentage and plant morphology measurements

Disinfected seed were sowed onto magenta boxes with MSagar- media, either alone as a negative control, supplemented with XGO at 0.1μM or 100mM NaCl to induce salt stress, or a combined (both NaCl+XGO). The concentration of the NaCl solution was determined based on experimental data (data not shown) in which it was considerable biomass decrease in the presence of 100mM NaCl was demonstrated. Each treatment consisted of nine seeds per magenta box and five boxes per each biological replicate. Germination percentage was calculated 10 days after sowing. Germination criteria were considered complete germination after the embryo emerged from the seed and a whole seedling was formed [30]. For plant morphology analysis, seedlings were grown for two months, and the number of leaves was then counted. Also, leaves were harvested by cutting three of the oldest ones to measure their foliar area, which were immediately photographed under a stereomicroscope (Olympus, Model CX31-RTSF) coupled to an Infinity Analyzer camera. FA was measured using the Infinity Analyze 3 software (Lumenera) according to the manufacturer instructions. For root length measurements and lateral root primordium frequency, sterilized seeds were plated on square Petri dishes in a vertical orientation containing MS-agar-media as control or supplemented with XGO at 0.1μM or 100mM NaCl to induce salt stress or a combination of XGO+NaCl. Each treatment consisted of six seeds per Petri square dishes and four dishes per each biological replicate. All measurements were performed on 3-week old plants that were fixed for 72h as previously described and had exhibited whole root systems [18]. The total number of lateral roots, which is the sum of the number of lateral root and number of primordia, were counted directly under a stereomicroscope (Olympus, Model CX31-RTSF). The fixed plants were then placed on a slide to allow the primary root extension and measurements by taking photographs and processing the images. A microscope (Olympus, Model SZ2-ILTS) coupled to an Infinity Analyzer camera was used, and primary roots lengths were measured using the Infinity Analyze 3 (Lumenera) software according to the manufacturer instructions. Lateral root density (DLR, represented as D in the formula) per mm of primary root length (PRL, represented as L in the formula) was calculated using the following equation: D = (RL+P)/L, where RL + P is the sum of the number of lateral root and number of primordia [31].

Induction conditions for biochemical analysis

For all of the biochemical analyses, a uniform induction experiment was designed in order to analyze XGO- (alone or in combination with salt shock) induced dynamic changes in N. tabacum seedlings. Specifically, we focused on the restitution phase (stage of resistance, continuing stress) of plants’ stress response phases [32]. Consequently, seeds were first placed in magenta boxes containing MS-agar-media to allow their homogeneous growth for 21 days. After that time, four treatments were administered:

a. One mL of sterile distilled and deionized water as a negative control.

b. A solution of 100 mM NaCl for salt shock.

c. 0.1μM XGO.

d. 0.1μM XGO+100mM NaCl were added to the top of the solid media.

Sampling leaves were taken at two and five days after induction, immediately frozen in liquid nitrogen, and stored at -80 °C until their use.

Quantification of proline and photosynthetic pigment content

After two and five days of XGO application with or without salt shock using NaCl 100mM, proline and total chlorophyll (Chl a+b) contents were measured. Proline extractions and quantifications were performed as previously described [33]. Briefly, the extract was prepared by mixing 20mg of ground leaves in 1mL of 80% ethanol, sonicated for 5min, and incubated for another 20min in the dark. The mixture was centrifuged at 20,000g for 5min, and 200μL of the supernatant were added to 400μL of reaction mix (ninhydrin 1% (w/v) in acetic acid 60 % (v/v), ethanol 20% [v/v]), and heated at 95 °C for 20min. Finally, absorbance was determined at 520nm using a FLU Ostar Omega Microplate Reader (BMG LABTECH GmbH, Germany). A calibration curve was used for proline concentration quantification and expressed as μg of proline per mg of plant fresh weight. For photosynthetic pigment content quantification, the Chl a and b and total chlorophyll (Chl a+b) contents were extracted with 80 % acetone and measured as described elsewhere [34].

Protein and lipid oxidative damage

Plant leaf tissue was ground to a fine powder with liquid nitrogen to ensure sample homogenization. Protein oxidation was measured using protein carbonyl content [35]. Briefly, 100μL of sodium phosphate buffer (PBS, pH 7.8) was added to 400mg of each sample, sonicated for 20min in the dark, centrifuged at 20,000g during 20min at 4 ºC, and the supernatant was used. Protein concentration in the supernatant was measured using a BCA Quantic Pro Sigma Kit to compare carbonyl content related to total protein content. Total carbonyl content was measured using dinitrophenylhydrazine (DNPH) reagent [35].

Lipid oxidation was analyzed with a thiobarbituric acid reactive substances (TBARS) method [36]. For the assay, 200mg of ground plant sample was submerged in 1000μL of acetone and sonicated for 5min. The mixture was then incubated for 10min in the dark and centrifuged at 4 °C and 20,000g for 5min. Then, 200μL of the supernatant was added to 300μL of a reaction mix containing 2:1 of 20 % (v/v) trichloroacetic acid (TCA) and 0.67% (w/v) of thiobarbituric acid (TBA). The reaction mix was heated at 95 °C for 15min, cooled at room temperature, and centrifuged at 20,000g at 4 °C for 20min. Lipid oxidation was measured by determining the absorbance at 532nm using a FLU Ostar Omega Microplate Reader (BMG LABTECH GmbH, Germany). The methylenedianiline (MDA) standard and standard curve for the estimation of total MDA were prepared as previously described [37]. Results were indicated as A532 per gram of plant sample.

Antioxidant enzyme activities

Plant leaves collected (0.4g) were homogenized in liquid nitrogen and 100μL of PBS (pH 7.8) containing protease inhibitor (Sigma Aldrich) concentration 5X. The mixture was then sonicated for 20 min in the dark and centrifuged at 20,000g at 4 ºC for 20min, after which time the protein content was measured using a bicinchoninic acid (BCA) Sigma® kit. Enzyme activities were determined immediately. Activities of the antioxidant enzymes, CAT (EC EC 1.11.1.6), GPX (EC 1.11.1.7), GR (EC 1.6.4.2, GR), and SOD (EC 1.15.1.1, SOD) were determined. The evaluation of enzymatic activities was performed by comparing equal amounts of total protein extracts from the samples collected.

CAT activity was measured by monitoring the enzyme-induced decomposition of an H2O2 solution at 240nm and calculated as H2O2 reduced per mg of protein per min [38]. GPX activity was assayed as previously described [39], in which the reaction mixture contained potassium phosphate buffer (100nM), guaiacol (15mM, pH 6.5), H2O2 0.05 % (v/v), and 60μL of protein extract. Guaiacol oxidation was monitored at 470nm and an enzyme unit was defined as the production of 1μm of oxidized guaiacol per mg of protein per min. SOD activity was measured by adapting the previously described chromogenic assay [40] for leaf tissue protein extract analysis. Briefly, for the reaction 225μL sodium pyrophosphate (pH 8.3, 0.025M), 18.8μL (186μM) phenazine methosulfate, 56.3μL (300μM) nitroblue tetrazolium, 93.7μL distilled water, and 5μL of protein extract were mixed. To initiate the reaction, 37.5μL (780μM) nicotinamide adenine dinucleotide was added, incubated for 1.5 minutes and 187.5μL glacial acetic acid were added to stop the reaction. The chromogen was extracted by addition of 700μL n-butanol followed by incubation for 10min, centrifugation at 20,000g for 5min, and absorbance measurement at 560nm. For GR activity measurement, the reaction was started by the addition of oxidized glutathione, and the decrease in absorbance at 340nm every min over a 3min period was read [41]. GR activity corresponded to the amount of enzyme required to oxidize 1μmol min-1 of nicotinamide adenine dinucleotide phosphate. For all enzyme activity analyses, results were expressed as U mg-1 protein.

Statistical analysis

For all variables analyzed, each experiment was performed in triplicate. The data were expressed as average ± standard deviation (SD) of the three independent replicates as a measure of dispersion. For the variable NL, FA, PRL, and DLR, the data were evaluated by an analysis of variance (ANOVA) by ranks (Kruskal Wallis test) and compared using a nonparametric multiple comparison test proposed by Conover [42] because the variables evaluated did not show a normal distribution and had heterogeneous variances. The adjustment to the premises was verified through the tests of Shapiro Wilk and Levene. PCA was performed with Pearson correlation matrices to represent a two-dimensional plane of treatment effects upon the five morphological traits [43]. The values of eigenvectors higher than the mean of the minor and the major values of the component were considered as significant.

Data obtained from biochemical analyzes were processed using a factorial ANOVA using a fixed effect model, in which the factors consisted of the treatments (XGO±NaCl) and the days after each treatment (two and five days). Previously, compliance with the normality and homogeneity premises were verified through the Shapiro-Wilk and Levene tests. All statistical analyses were performed with the InfoStat program [44].

Resultst

Effect of XGO on germination and growth of Nicotiana

The effects on GP and plant and root growth of N. tabacum seedlings, grown with MS ± 0.1μM XGO, salt stress with ± 100mM NaCl, or a combination of XGO and NaCl are shown in Table 1 and Figure 1. GP was statistically similar between the negative control and 0.1μM of XGO (close to 90%). However, GP was significantly reduced to 71% and 75% with 100 mM NaCl and 0.1μMXGO+100mM NaCl, respectively, compared to the MS negative control (P<0.05). Both salt stress control (MS+NaCl) and XGO+NaCl were statistically similar. On the other hand, 0.1μM XGO significantly caused a promotion in FA and PRL in two-month-old N. tabacum seedlings compared to negative control, but no statistical differences were observed in NL or in DLR. Moreover, when XGO was combined with 100 mM NaCl, there was a significant increase in NL, PRL, and DLR (P<0.05) related to salt control, but no statistical differences were observed in FA (Table 1 & Figure 1). Thus, addition of 100 mM NaCl caused an inhibition of NL and PRL by 36.3% and 43%, respectively, in N. tabacum seedlings compared to the MS negative control. Nevertheless, the inhibitory effects of salt stress on NL and PRL were reduced to 16.5% and 15.4%, respectively, compared to untreated control when XGO was incorporated in the media.

MS: Negative control; XGO: 0.1μM); MS+NaCl: Salt stress with 100mM NaCl; XGO+NaCl: 0.1μM XGO + 100mM NaCl; GP: Germination percentage; NL: Number of leaves; FA: Foliar area (mm2); PRL: Primary root length (mm); DLR: Density of lateral root.

The PCA explained the contribution percentage of each component to the total variation with the five quantitative characters evaluated (GP, NL, FA, PRL, and DLR) and each treatment (XGO and/or salt stress) (Table 2). The first two principal components (PC 1 and 2) justified 99.3 % of the total variation. The characters with the greatest contribution to variability consisted of NL, FA, and PRL in PC 1, and DLR and GP in the PC 2. The biplot chart of first and second component showed that PRL and DLR were significantly correlated (P<0.05) in addition to NL, FA, PG, and NL with PRL (Figure 2). There is a separation between treatments with NaCl either with or without XGO application in the first principal component, and in component 2 there was a clear separation of XGO treatments from those without XGO. The higher values in PRL, DLR, and NL correspond to treatments where XGO has applied alone, compared to those where it was combined with salt stress. In the second component, DLR and GP characters showed the highest positive and negative contribution, respectively. Also, there was a well-defined separation of XGO and XGO+NaCl from MS and MS+NaCl, with the higher average DRL seen with the XGO values.

Changes in proline and total chlorophyll content

Figure 3 shows the effect of XGO and salt stress on proline and chlorophyll contents of N. tabacum leaves measured after two and five days of induction. It can be noticed that treatments without salt (MS±XGO) exhibited no significant differences in proline content after two and five days of treatment (Figure 3a). Additionally, salinity stress in N. tabacum seedlings promoted significant proline accumulation compared to untreated control at both time points. However, the results obtained XGO+NaCl application reflects a gradual and significant increase in the proline content, which is 75.98% higher than salt stress control after five days of treatment (Figure 3a).

Total chlorophyll (Chl a+b) content was significantly higher after XGO application compared to the untreated control, which reached the highest levels among all treatments at both time points (Figure 3b). Also, as expected the Chl a+b content was significantly reduced by salinity stress in N. tabacum leaves compared to the untreated control and remained constant over time (P<0.05). On the other hand, XGO combined with NaCl produced a significantly higher Chl a+b content (29.58% upper) compared to salt stress control after five days of treatment, which reached levels similar to the negative control.

Changes in protein and lipid oxidative damage

The effects of XGO and salt stress on protein oxidation, measured as total carbonyl contents, in N. tabacum leaves at two and five days after induction is shown in Figure 4a. Plants treated with XGO exhibited no significant differences in total carbonyl content related to control plants after two and five days of treatment. On the other hand, NaCl application significantly increased its content by 67.10% compared to the untreated control after five days. In contrast, at the same time point, XGO application combined with salt stress significantly reduced protein oxidation in N. tabacum leaves by 98.45% compared to the salt stress control.

Lipid peroxidation was calculated in terms of MDA content as an indicator of lipid oxidative deterioration caused by severe oxidative stress in N. tabacum leaves at two and five days after induction (Figure 4b). XGO application caused the MDA amounts to remain constant over time. On the other hand, leaves from plants exposed to salinity stress demonstrated significantly higher MDA accumulation compared to untreated control at both time points. However, XGO application and salt stress also significantly caused a reduction in lipid peroxidation by 51.88% compared to salt stress control after five days of treatment.

Changes in activities of enzymes from the antioxidant system

We also examined four oxidative stress response enzyme markers in the context of their activities. Figure 5 shows the effect of XGO application and salt stress on the activities of the antioxidant enzymes, CAT, GPX, SOD, and GR in N. tabacum leaves after two and five days of induction. The results showed that five days after treatment with XGO alone resulted in a significantly higher CAT, GPX, and SOD activities over time compared to the negative control (P <0.05) (Figure 5a-c). GR activity was not significantly affected by the application of any treatment (Figure 5d). Salt shock with 100mM NaCl induced significantly higher CAT, GPX, and SOD activities two days after induction compared to the negative control. After five days in the presence of NaCl, GPX decreased, but CAT activity remained higher (P<0.05) than the untreated control. There was also an apparent increase in CAT, GPX and SOD activities after five days of treatment with XGO+NaCl, but their levels were statistically similar to those of salt control (Figure 5a–c).

Discussion

This study provides evidence concerning the effects of exogenous application of XGOs on enhancement of N. tabacum growth and development under saline conditions and salt shock in order to allow them to overcome the salt-stress limitations. Clearly, under the conditions used in the current work, XGO alone had a positive effect on NL, FA, and PRL although it was ineffective in promoting DRL. However, combined with continuous salt stress, this effect is rearranged, and XGO causes an increase in NL and PRL in addition to promoting lateral root formation although no changes in FA were observed compared to the salt control. Consequently, in the presence of XGO the plants managed to recover from limitations imposed by salt, so they display visible morphological characteristic improvement (Figure 1).

The analysis of eigenvalues corresponding to morphological changes is based on PCA analysis and explains >99% of the total experimental variability within the two first components. This is almost 100 % of the experimental variability that was achieved by reducing up to two principal components. The five evaluated traits showed a high contribution in one of the two first principal components; thus, each trait was very important to explain the variability observed in the experiment. PC 1, which explains the highest percent of variability (90.7%) allowed for separation of treatments under salt stress (100mM of NaCl) from those without NaCl. The highest values of NL, PRL, and FA were obtained in the medium with XGO and without salt stress, which were projected in the positive quadrant of the component. This result clearly indicates that XGO in the medium enhanced PLR and increased FA and NL in tobacco seedlings. PC 2, which explains the rest of the variability (8.6%), separates the treatments with XGO from the treatments without this biostimulant. In this component, the traits that showed the highest contribution were PG and DLR. According to these findings, the presence of XGO in the medium stimulated DLR, but the highest values of PG were obtained in the MS medium without NaCl.

These results confirmed that external application of 0.1μM XGO positively influenced plant growth and morphological features even under salt stress conditions and could be correlated with auxin-like activity. It is known that at approximately 1μM, at least four different cellotetraose-based XGOs (XXXG, XXLG, XXFG, and XLLG) mimic auxin by inducing growth [45]. In a previous study, we confirmed that auxin-like activity of the same XGO fraction mix and concentration used in this work on Arabidopsis thaliana seedlings [18]. These are important findings since the ability of a biostimulant to influence plant hormonal activity is one of their many important benefits because they can exert large influences that eventually will improve their health and growth. As plant growth regulators plays an important role as chemical messengers, they alert the plants when stressful environmental conditions exist so they can initiate or increase their stress response processes [46].

In this context, XGO may be acting as a “switches” that turn on the plants for stressful situations by altering hormonal balances. Zhang and Schmidt (1999) discuss the “switch” concept and give some examples of other types of biostimulants that reinforce our evidence. In this context, the results also suggested that exogenous XGO applications could be acting as “pre-stress conditioners” [46] and their effects are manifested by improving osmotic regulation, photosynthetic efficiency, or by causing an increase in antioxidant levels. This argument is based on the results in which it was shown that 21-day-old plants exposed to external application of 0.1μM XGO had higher proline accumulation and total Chl content in addition to higher CAT activity levels, GPX, and SOD compared to untreated plants. Of more interest was their effect when applied in combination with salt shock (XGO+NaCl) compared with those treated with NaCl alone (MS+NaCl). In this case, the highest recorded proline levels in addition to higher total Chl were observed after five days of treatments. Enhancement of this antioxidant machinery could be reflected in significant protein oxidation (total carbonyl content) and lipid peroxidation reduction. Therefore, it can be seen that the XGOs help the plant to cope with the effect of saline stress via proline accumulation.

Osmotic regulation is an important mechanism for plant cellular homeostasis under saline conditions in which proline is the most common osmolyte for osmoprotection [47]. The higher accumulations of proline recorded with XGO and NaCl at five days after treatment could be correlated with stress tolerance and may participate in the stress signal influence on adaptive responses (Figure 2) [6]. Proline also contributes to stabilization of sub-cellular structures (such as membranes and proteins), and its cytoplasmic accumulation could help reduce oxidative stress-generated plant protein and membrane damage after exposure to salinity [6]. This effect can be inferred because of lower total carbonyl content levels since protein-bound carbonyls represent a marker of global protein oxidation and lipid peroxidation products as biomarkers for oxidative stress that were observed in plants treated with XGO+NaCl (Figure 3). Therefore, XGO indirectly helps the cell cope with salt stress by maintaining cellular osmotic adjustment and protein and lipid integrity.

Another important result indicated that XGO seems to mitigate negative effects on photosynthesis in stressed plants by increasing Chl content. This parameter was used because of salinity- induced increase in chlorophyllase activity with the consequent degradation of chlorophyll (at least transiently). Consequently, we can deduce that the increase in exogenously XGO-induced total Chl content enabled tobacco plants to tolerate salt-stress in addition to promoting their development and growth. Similar results with photosynthetic pigments were obtained in our lab when the same XGO fraction mix was evaluated on A. thaliana seedling growth under saline stress [48].

Regarding the activity of antioxidative enzymes, exogenous application of 0.1μM XGO increased CAT, GPX and SOD enzyme activities compared to the negative control after five days of treatment. Due to the fact that no significant XGO+NaCl effects on enzymes’ activity that were analyzed in this work were observed, it would be advisable to analyze other antioxidant enzymes to expand the analysis in addition to determining its mode of action in the enzymatic antioxidant system.

Our study revealed that XGO seem to be working as metabolic inducers that trigger the physiological responses mentioned above. Some results support that xyloglucan fragments do not penetrate the cell, but instead, it has been suggested that the existence of specific receptors on the plasma membrane, which interact with the fragments, activate a signaling cascade inside the cell [49]. However, to date, no specific candidate has been identified as a possible receptor of these molecules [19]. Also, it has been suggested that they can promote modifications or integrate into the cell wall, which can affect not only the extracellular events in the wall but also intracellular events [50]. These authors demonstrated that incubation of pea stem segments partially bisected longitudinally with a xyloglucan oligosaccharide (9mM XXXG), accelerated the cell elongation by integration of xyloglucans as they were incorporated into the cell wall and became transglycosylated by xyloglucan endotransglycosylase (XET). According to this, XGO’s effects observed in our work may also result from a signal transduction XET-mediated or -induced cell wall modification cascade but not from the oligosaccarides’ direct actions.

Conclusion

Overall, we conclude that XGO can exert beneficial impacts on tobacco plants’ stress response either through hormone-like effects, osmotic regulation, photosynthetic efficiency improvement, and increase in antioxidant levels. They promote the proline accumulation as an organic osmolyte and increase total chlorophyll content and modify some antioxidative enzymes’ activities that eventually affect development of plant roots’ growth and development under salt stress. Further in vivo studies are needed to confirm the antioxidant effect of XGOs during salt stress in crop plants as well as to unravel their mechanism of action on oxidative responses. However, according to these results, the exogenous application of XGO as biostimulant at very low concentrations could be considered an alternative for improving the growth and productivity of crops of agronomic importance under salt stress.

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