Genotype-specific Patterns of Physiological, Photosynthetic, and Biochemical Responses in Faba Bean Contrasting Pair to Salinity

Abstract

To understand the salinity tolerance mechanism in faba bean contrasting pair to salinity (cv. Chourouk as sensitive and cv. Najeh as tolerant), we evaluated the effect of high salt concentration (150 mM NaCl) on the photosynthetic, physiological, and biochemical parameters at short and long term of treatment (1 and 6 days, respectively) in the seedling stage. In general, the salinity affects the growth of plants. High salinity decreased all studied parameters, especially transpiration rate (E), stomatol conductance (gs), net CO2 assimilation (A), and substomatal CO2 concentration (Ci), and dramatic changes was registered in cv. Chourouk compared to cv. Najeh. Chlorophyll contents were also affected by salinity, especially in the sensitive variety. In addition, the synthesis of osmolytes (proline) was determinate, to understand whether the osmotic adjustment is a mechanism used by cv. Najeh to tolerate salt stress. Our research suggests that cv. Najeh should be introduced in a crossbreeding program as an elite salt-tolerant germplasm.

1. Introduction

One of the most damaging abiotic stimuli that affects crop output and changes in plant growth and development is salt stress. High salinity is thought to affect 20% of the world’s arable land and 33% of its irrigated agricultural land, with the total area affected by salinity growing at a pace of 2% per year [1]. Additionally, it has been predicted that by 2050, salinity will affect more than 50% of the entire area currently used for agriculture [2]. In semiarid and arid locations, where other environmental stresses are more prevalent, the effects of salt stress are more severe. For example, insufficient precipitation can create drought, heat can produce excessive surface evaporation, and irrigation with saline water can exacerbate salinity problems [3]. In response to salt stress, various elements of plant development at the germination, vegetative, and reproductive stages are altered because of interactions between physiological, morphological, and biochemical systems [4]. The plant is subject to nutritional deficiency, oxidative stress, ion toxicity, and osmotic stress because of the soil’s salinity. The main two reasons of the growth inhibition of salinity were as follows: the high concentration of salt which decrease the ability of plants to adsborb water, and this can lead to the touble in the growth rate (water-deficit effect or osmotic effect); and the ionic effects which due to the diffusion of high amounts of salt in the plant tissues will cause the damage of the cells [5]. However, the level of salt tolerance differs amongt species [6].

The flowering plant Vicia faba, commonly known as the broad bean, fava bean, or faba bean, horse bean, field bean, bell bean, Windsor, or tic bean, is native to North Africa and south-western Asia and is widely cultivated abroad [7, 8]. In warm temperate and subtropical regions, it is grown as a winter annual. While the hardiest European cultivars may withstand winter temperatures as low as −15°C, harder cultivars growing in the Mediterranean region can withstand winter temperatures as low as −10°C [9]. Due to its high protein content, which can range from 20 to 41% depending on variety, faba bean seeds are particularly essential crops [10]. Additionally, they have enough amounts of both carbohydrates and oil, which raises their nutritional value [10]. Due to their great nutritional content and positive health impacts, the consumption of legumes has recently expanded around the world [11, 12, 13, 14].

Two types of faba beans, Vicia faba L. var. major and Vicia faba L. var. minor, are grown in Tunisia, and on average, faba bean cultivation takes up roughly 68 percent of the country’s total acreage for growing grain legumes area [15]. The average of dry seed yield of the country is 0.99 t ha⁻¹, which is less than the globally average yield (1.7 t ha⁻¹). However, the production of faba beans in Tunisia varies from year to year because of the lack of cultivars that are resistant to biotic and abiotic challenges (mostly salt). The creation of salt-tolerant cultivars is a crucial method for reducing the detrimental impact of salinity on agricultural productivity. Crop tolerance to salt has long been studied using plant breeding techniques and traditional selection methods. It has been noted that selection based on physiological characteristics can ameliorate salt tolerance in crops better than selection based on agronomic traits [16]. Based on the physiological aspects, Rajhi and his collegues [8] succeed to select a pair of Vicia faba with contrasting behaviour to salinity stress; cv. Chourouk was selected as sensitive cultivar and Najeh was considered as a tolerant one [7, 8]. Thus, the objective of this study is to study the genotype-specific patterns of physiological, photosynthetic, and biochemical responses in faba bean contrasting pair to salinity, grown under high salinity level (150 mM of NaCl).

2. Materiels and methods

2.1 Plant materials

Two Tunisian faba bean cultivars (cv. Najeh and cv. Chourouk) were chosen in this study to understand the physiological and biochemical tolerance to stress salinity. Seeds were offred from the Field Crops Laboratory of the National Institute of Agricultural Research in Tunisia (INRAT).

2.2 Growth conditions

This study was conducted in the greenhouse of the Biotechnology Center of Biotechnology of Borj Cedria under controlled conditions: 23°C, 16/8-h day/night photoperiod, relative humidity of 55–65%, and photosynthetically active radiation of 270 μmol (photon) m⁻² s⁻¹. Seeds were first surface sterilized by soaking them in a solution of 0.1% mercuric chloride (HgCl₂) for 1 minute, followed by a thorough rinsing with sterilized distilled water. They were then planted in humidifying perlite at room temperature (20°C) in the experimental field for 7 days. Then, similar size seedlings were moved into plastic pots filled with a half-strength nutritional solution for 7 days. After that, the seedlings were transferred to a full-strength nutrient solution containing micronutrients (in μM: CuSO₄, 1.56; ZnSO₄, 1.55; H₃BO₃, 4; (Na)₂MoO₄, 0.12; MnSO₄, 6.6; CoSO₄, 0.12) and macronutrients (in mM: K₂SO₄, 0.7; KNO₃, 24; KH₂PO₄, 0.36; CaCl₂, 1.65; MgSO₄, 1) [17]. Every week, fresh nutrient solutions were added, and a hydroponic air pump system continuously aerated them at 400 ml/min.

2.3 Salt stress treatment

Plants were subjected to salinity treatment when they reached the four-leaf stage under two different settings: (1) control conditions (0 mM NaCl) and (2) severe salt concentration (150 mM NaCl). By gradually introducing 25 mM of NaCl into the nutritional solution each day, salinity stress was induced. Weekly nutrient solution changes were made to both the treated and control plants. The treated and control plants were both collected individually. Nine replications were considered for each cultivar per treatment.

2.4 Measurements of morphological parameters

The number of leaves, shoot lengths, and root lengths were the morphological parameters that were measured in this study. The distances between the crown and the leaf tip (in cm) and the crown and the root tip (in cm), respectively, were used to calculate the lengths of the shoots and roots. Counting was used to establish how many leaves there were.

2.5 Plant biomass

Treated and control plants were harvested and divided into roots and shoots. On the day of harvest, the weights of the shoots and roots were measured. Each part was dried at 70°C until a constant weight was attained, and then the dry weight was measured.

2.6 Relative water content

According to Barrs and Weatherley’s instructions [18], the relative water content (RWC) was calculated. To determine the fresh weight, the topmost fully expanded leaves were weighted (FW). The leaves were then weighted again to obtain the turgid weight after being immersed in distilled water for 24 hours (TW). Following that, the samples were dried at 70°C for 72 hours, and the dry weight was calculated (DW). The following equation was used to calculate the RWC: RWC (%) = {(FW∕DW)∕(TW∕DW)} *100.

2.7 Gas exchange measurements

The photosynthetic parameters were determined using the youngest fully developed leaf under the following conditions: full sunlight and at 10:00 a.m. under atmospheric CO₂. An open-type and portable photosynthetic system (LC pro+; Bio-Scientific, Great Amwell, Herts, UK) was used to monitor the internal concentration of CO₂ (C_i), stomatal conductance (g_s), net CO₂ assimilation rate (A), and transpiration rate (E).

2.8 Chlorophyll content

Using the Lichtenthaler method [19], the chlorophyll content in different samples was estimated. Young leaves that had just been cut (100 mg) were foated in 5 ml of an acetone solution (80% of acetone) at 4°C and in the dark until the chlorophyll extraction process was complete. The amount of total chlorophyll was determined by measuring absorbance (A) at 645 and 663 nm using a UV-visible spectrophotometer (Jenway 6850 UV–Vis; ColeParmer Ltd., UK). The chlorophyll content was measured as mg g−¹ as following: total chlorophyll content = 6.45 × A663 + 17.72× A645.

2.9 Electrolyte leakage

According to Dionisio-Sese and Tobita [20], electrolyte leakage (EL) was calculated. Freshly cut leaves were placed in an assay tube containing ultrapure water, and the tubes were then incubated at 32°C for 2 hours. During this time, the solution’s first electrical conductivity (EC1) was measured using a Metrohm 712 conductometer (Metrohm AG, Herisau, Switzerland). Following the EC1 measurement, the same tubes were incubated in a 90°C oven for 2 hours. Then, the solution was cooled to 25°C, and the second conductivity (EC2) measurement was performed. Electrolyte leakage parameter was calculated using this formula: EL = EC1/EC2 × 100.

In total, 15 parameters were measured (Table 1).

Number	Parameters	Abbreviation
1	Root length	-
2	Shoot length	-
3	Root fresh biomass	-
4	Shoot fresh biomass	-
5	Root dry biomass	-
6	Shoot dry biomass	-
7	Leaf number	-
8	Proline	-
9	Relative water content	RWC
10	Chlorophyll	-
11	Electrolyte leakage	-
12	Transpiration rate	A
13	Stomatal conductance	gs
14	Net CO2 assimilation	A
15	Stomatal CO2 concentration	Ci

Table 1.

Different used parameters.

In the current investigation, all morphological and physiological data were converted into salt tolerance indices using the method of Zeng et al. [21] and Rajhi et al., [8]. These indices were calculated by dividing the salinity-related observed value by the control averages. The difference between treatment data was estimated using the STATISTICA software and the means of comparison by HSD (higher significant difference) Duncan’s test (p ≤ 0.05).

3. Results

In the current study, 15 physiological, morphological, and biochemical characteristics were used to compare the responses of a contrasting pair of faba bean genotypes to severe salt concentration (150 mM) as well as control conditions.

All the data were converted to relative values, or salt tolerance index. The observation under salinity was divided by the means of the controls to calculate the salt tolerance index. The treated and control plants were grown in identical environmental conditions and harvested simultaneously with and without the addition of NaCl, respectively.

3.1 Effect of salinity on plant length, fresh and dry biomass, and leaf number

Figure 1 describes the effects of salinity stress on the growth characteristics of faba bean plants. In general, this figure shows a significant difference between both cultivars during treatment time. Seedling shoot and root lengths of cv. Chourouk decreased with the application of stress after 6 days compared to 1 day (Figure 1a). However, the length of the root of tolerant cv. Najeh did not affect by salinity applied for short or long time compared to the other cultivar. Figure 1b clearely demonstrated that the fresh root biomass of cv. Najeh had the highest value compared to root and shoot of cv. Chourouk at short time of stress application. Nevethless, after 6 days of stress conditions, cv. Najeh exhibited the lowest fresh biomass compared to shoot and root of cv. Chourouk. Furthermore, the dry biomass of cv. Najeh presented the highest value compared to the root of cv. Chourouk after short or long time of stress application (Figure 1c). On the other hand, the dry biomass of shoot of cv. Chourouk exhibited the lowest value after 6 days of NaCl supply.

Figure 1.

Index of tolerance of root and shoot lengths (a), index of tolerance of fresh root and shoot weights (b), index of tolerance of dry root and shoot weights (c), and index of tolerance of leaf number (d) of cv. Najeh and cv. Chourouk. All values are means ± SD. The data followed by different letters are significantly different at p ≤ 0.05.

The results of the number of leaves shown in Figure 1c indicate that the use of high salinity concentration decreased the number of leaves in stressed plants compared to control ones. Cv. Chourouk had the lowest tolerance index of leaf number compared to cv. Najeh, which presented the highest values after 1 or 6 days of salt treatment.

3.2 Effect of salinity on proline content, total chlorophyll content, electrolytes leackage, and RWC

Proline accumulation is an important mechanism for osmotic regulation under salt stress. In this study, we evaluated proline accumulation profiles in roots and shoots of a contrasting pair of Vicia faba to salinity stress (Figure 2a). An increase in proline accumulation was increased in all plant’s parts. Shoot and root of cv. Najeh exhibited the highest proline contents either at 1 day or 6 days of salt application. An important accumulation of proline was registered in the root of cv. Najeh after 6 days of tretement compared to cv. Chourouk.

Figure 2.

Tolerance index of proline content (a), RWC (b), total clorophyll content (c), and electrolyte leackage (d) of cv. Najeh and cv. Chourouk. All values are means ± SD. The data followed by different letters are significantly different at p ≤ 0.05.

Figure 2b illustrates the impact of salinity on RWC. A significant difference was found between both cultivars in response to 150 mM of salt concentration. The index of tolerance of RWC of cv. Najeh was not affected by high salinity stress either after 1 or 6 days of treatment. The highest RWC was recorded for cv. Najeh compared with cv. Chourouk.

The chlorophyll content of the leaves of the faba bean cultivars significantly decreased because of exposure to the important amount of NaCl, as evidenced by the results in Figure 2c. After 24 hours of salt application, cv. Najeh exhibited the highest value in term of chloroplyll content compared to cv. Chourouk. However, after 6 days of treatmet, both cultivars exhibited the same value of chlorophyll.

In this study, we noted a significant increase in electrolyte leackage in stressed plants (Figure 2d). The electolyte leackage increased with the increase of the time of treatment. The best results were observed in cv. Najeh after short and long time of treatment compared to cv. Chourouk, which had the highest value.

3.3 Effect of salinity on photosynthetic parameters

Figure 3 displays the effects of salinity on the leaf gas exchange parameters in faba bean cultivars under treatment condition during different time. In comparison to their respective controls, the index of tolerance of Ci of both cultivars did not change after 1 day of salt application, and they exhibited a comparable value (Figure 3a). However, after 6 days this parameter was affected by salinity and a large decrease was detected for cv. Chourouk. A significant decrease was also observed for E parameter for both cultivars (Figure 3b). In addition, this parameter was lower after 6 days compared to 1-day salinity duration. A significatant fluctuation was recorded between both cultivars, and cv. Najeh showed the highest E value compared to cv. Chourouk. No signifcant diference was found in g_s values between cultivars (Figure 3c). Neverthelees, long salt period affects this parameter. The A parameter was also affected by salinity (A), especially after 6 days of treatment (Figure 3d). However, we did not find a significant difference between both cultivars.

Figure 3.

Tolerance index of internal concentration of CO₂ (a), transpiration rate (b), stomatal conductance (c), and net CO₂ assimilation (d) of cv. Najeh and cv. Chourouk. All values are means ± SD. The data followed by different letters are significantly different at p ≤ 0.05.

4. Discussion

The number of regions with salt-affected soils is likely to grow in the future years, especially for arid and semiarid regions like Tunisia, which is facing the most severe impacts of salinity stress. A rapidly growing global population will have fewer food options due to limited crop output caused by the degradation of fertile land. The creation of salt-tolerant plants may make it possible to cultivate saline-affected land. The current study was conducted in this context to establish morphological and physiological characteristics that can be used to assess the tolerance of faba bean to salinity and to compare the salt tolerance of two faba bean cultivars with contrasting behaviour to salinity at the seedling stage. Our results showed that salinity negatively affect the lengths, the fresh, and dry biomasses of plants. These results reveal that salinity treatment had an impact on the study plants’ growth and biomass development, which are in accordance with the data provided by Tavakkoli et al. [16], Hashem et al. [22], and Dawood and EL-Awadi [23]. This decrease in growth and biomass is caused by the suppression of cell extension and division, production of reactive oxygen species, decrease in mineral intake, hormonal imbalance, and inhibition of enzyme activity [24, 25, 26]. NaCl harmful effects on plant metabolism, particularly sensitive plants, cause their slowly growth. It has been discovered that sensitive cultivars are more susceptible to salinity than tolerant ones [24]. Salinity treatment significantly decreased the root and shoot lengths and weights of the cv. Chourouk compared to cv. Najeh. This pattern can be a sign of salt sensitivity. Our findings demonstrate that faba bean plants lost leaves when exposed to salnity stress. Similar results have been reported in diferent plants [27, 28, 29]. Our findings demonstrate that cv. Najeh was able to maintain the number of leaves under salinity circumstances compared to control ones, since it exhibited the smallest decrease in leaf number at short and long term of salt treatment.

Different physiological reactions and changes in photosynthetic mechanisms cause the reduction of plant growth that occurs under salt conditions. Stomatol closure caused on a decrease in intracellular CO₂ is thought to be the cause of photosynthetic limitations during salt stress [30]. Our experiment demonstrated that salinity stress significantly affected plant growth by inhibiting A, E, g_s, and C_i parameters. Our results agree with those reported by Mohamed et al. [31] and Alzahrani et al. [32]. Cv. Najeh exhibited less reduction in photosynthetic activity compared to cv. Chourouk, when grown under 150 mM of NaCl. This stability of photosynthetic parameters reflects an ability to maintain photosynthetic capacity and a better tolerance to salinity conditions. Cv. Najeh is considered as a tolerant cultivar, because it exhibited a better photosynthetic performance than the sensitive one.

Relative water content is the most relevant measure of the status of plant water under stress [33]. Cv. Najeh showed a stable and similar value of RWC after 1 or 6 days of treatment, and that may be due to the capacity for water absorption and water status and leaf hydration [34].

The plasma membrane is the main location of ion-specific salt damage [35]. The electrolyte leackage is considered as one of the criteria in the identification of tolerant cultivars to salinity [36]. In this study, the electrolyte leackage was slightely affected in cv. Najeh. However, cv. Chourouk recorded an increased value of this parameter. The same increasing trend was observed in salt-sensitive plants compared to tolerant ones [37].

Proline is the most prevalent endogenous osmolyte that accumulates under different abiotic stresses, such as salinity [38, 39]. It is widely known that specific exogenous proline concentrations control several aspects of plant growth and development under salt stress, such as increases in biomass and productivity [40, 41, 42]. When applied as an exogenous compound to crops, proline can improve salt tolerance [43]. Thus, the tolerance of cv. Najeh may be due to the accumulation of proline as osmolyte under salinity stress.

5. Conclusions

In conclusion, the physiological and photosynthetic parameters evaluated in this study demonstrated significant genotypic variation, confirming that variables that really do may be utilized as salinity tolerance screening criteria for faba beans. The examinated cultivars varied significantly in how they responded to salinity stress. However, the physiological characteristics of two cultivars were affected by salt stress to varied degrees, suggesting that these cultivars’ resistance to salinity varies. As a result of its capacity to preserve both its photosynthetic system and biomass, our data collectively indicate that cv. Najeh is the best cultivar in surviving salinity-stressed circumstances. The major distinguishing characteristics used to categorize faba bean varieties are thought to be photosynthetic and biomass parameters. The findings of this study demonstrate that several pathways contribute to salinity tolerance. This study specifically demonstrate that cv. Najeh can be classified as a salt-tolerant cultivar, which may be of great interest in future breeding projects for modern cultivar improvement. Finally, our research suggests that cv. Najeh should be introduced in a crossbreeding program as an elite salt-tolerant germplasm.