Impact of Severe Salt Stress on Morphological, Physiological, and Biochemical Parameters in Alfalfa (Medicago sativa L.) ^†

Abstract

In the Mediterranean area, climate change induces an important increase in soil salinity, which threatens crop production. Here, we aimed to test the impact of severe salt stress on the Alfalfa (Medicago sativa L.) used by Moroccan breeders. Stress was applied by increasing salinity gradually by 8 dS/m. Two weeks later, the root dry weight (RDW) and shoot dry weight (SDW) were measured as morphological parameters. Soluble proteins, total soluble sugars (TSS), and proline contents were quantified in both of leaves and roots parts. Furthermore, Chlorophyll a (Chl a), b (Chl b), and total Chlorophyll (Chl T) contents were quantified in leaves to estimate the salt stress impact on the photosynthetic apparatus. Our results showed a significant decrease in morphological parameters under stress. Additionally, a significant reduction in photosynthetic pigment was recorded. Compared to leaves, important accumulations of proline and soluble protein contents in roots were observed. These results indicate that under salt treatment, alfalfa plants react to salinity by storing many molecules in the roots for planned mobilization after recovery.

1. Introduction

Saline soils cover more than 7% of the world’s total land and 70% of all agricultural soils in the world. They have devastating global consequences and are projected to cause 30% land loss within the next 25 years and up to 50% by 2050 [1]. Unfortunately, in Morocco, there is a significant area of land affected by salinity, and about 500,000 hectares are reported to be affected [2]. Salinity is becoming one of the major environmental factors reducing crop yields and threatening the world food balance [3]. To overcome this environmental constraint, different strategies can be adopted, but these methods are very expensive. [4] Therefore, the introduction of resistant varieties to abiotic stress with a high socioeconomic value constitutes one of the approaches to rehabilitating saline soils. Selecting ideal plants for these conditions is the first step to solving the problem of salinity. [5] The objective of this study was to screen the effects of salt stress (induced by NaCl) on physiological and biochemical parameters in alfalfa (Medicago sativa L.).

2. Material and Methods

Homogenous seeds of alfalfa were disinfected and sown in pots containing approximately 1 kg of substrate (soil/peat/sand, (1:1:1)). Twenty-one days after sowing (DAS), two homogenous plants were maintained per pot, and salt stress was applied by irrigation with NaCl solution to increase soil conductivity to 8 dS/m. All pots were arranged randomly in greenhouse at 21 °C under natural light supplemented with artificial light with PAR of 300 µmol photons·m⁻²·s⁻¹. At 35 DAS, root and shoot parts were harvested, dried at 70 °C for three days, and weighted to estimate root (RDW) and shoot (SDW) dry weight. For physiological parameters, the Chlorophyll (Chl a), b (Chl b), and total Chlorophyll (Chl T) contents were determined using Burnison’s method (1980) [6]; total soluble sugars (TSSs) content was determined according to Erice et al.’s (2007) method [7]; proline was determined using Bates et al.’s (1973) method [8]; and soluble proteins content was quantified following Lowry’s method (1951) [9].

3. Results and Discussion

Our results show that salinity markedly affects all parameters studied. Thus, for morphological parameters, a significant (p < 0.05) reduction in RDW and SDW was recorded under salt stress compared to control plants (Figure 1). The reduction was 77% and 42% in roots and shoots, respectively. These results confirm what was previously reported in the literature [10,11]. This biomass reduction indicates that plants are trying to manage their resources by downregulation of their vegetative growth [12]. The measure of photosynthetic pigments content in leaves showed that salt stress significantly reduces Chl a, Chl b, and total Chlorophyll contents (Figure 1). The decrease in photosynthetic pigment under stressful conditions is widely spread in the literature [13], and it is reported to be linked to other metabolism disturbances, such as electrolytes leakage from thylakoids, the dehydration of protoplasm, lowered photo-assimilation levels, and Chlorophyll photo-oxidation [14]. Moreover, the decrease in photosynthetic pigment under stress is considered a sign of sensitivity [15]. An analysis of variance (

Table 1. Analysis of variance for proline and soluble sugar and protein contents in root plant part and area plant part (Df: degree of freedom. *: significant at 0.05, **: significant at 0.01. ***: significant at 0.005.).

Source	Df	Proline	TSS	Proteins
Part of plant (pp)	1	0.89142 *	29.8207 ***	246.385 ***
Treatment (trt)	1	5.38135 ***	64.6927 ***	399.661 ***
Replicates	2	0.05616	0.2490	1.208
pp*trt	1	0.74541 *	6.4557 **	20.663 *
Error	6	0.08834	0.4190	1.759
Total	11

) showed that both parts of plant and treatment factors and their interaction significantly influenced (p < 0.05) the parameters studied. The treatment factor is the most important source of variability for proline, TSS, and soluble protein contents, where this factor explained 75%, 64%, and 60% of the total variability, respectively. Moreover, part of plant factors explained 37%, 29%, and 12% of the total variability of soluble proteins, TSS, and proline contents, respectively, while the effect of the interaction between the last factors was less than 10% for all parameters studied. The results in Figure 2 showed a significant increase in biochemical parameters studied resulting from NaCl treatment in both leaves and root parts, which is in agreement with other studies [16,17]. A significant increase in soluble protein content was shown in salt treatment compared to the control in both leaves and root parts. Soluble proteins contribute to the osmoregulation process in indoor plant mediums [15]. Furthermore, significant increases in proline content in both root and shoot parts were recorded. The increase in proline in stressful conditions is well noted in various studies [11,16]. This amino acid is involved in many physiological functions, such as osmotic adjustment, free radical scavenging, cellular redox potential buffering, and detoxification pathway activation [18]. In alfalfa plants, the accumulation of proline salt stress is linked to salt tolerance [11].

The accumulation of soluble proteins and proline under salt stress appears to be more important in roots, which is also noted in many other papers [19]. It is widely known that alfalfa roots accumulate many molecules under stressful conditions to ensure a stock of molecules for mobilization after recovery [15]. Total soluble sugar contents also increased in stressed plants by 41% and 31% in leaves and roots, respectively. It is well documented that salinity increases the production of many sugars in alfalfa leaves, such as sucrose and pinitol [20]. These carbohydrates are considered osmoregulators that decrease the osmotic potential in order to maintain osmotic homeostasis inside of the cell [21]. The lower accumulation level of these molecules in roots compared to leaves might be explained by the storage of these molecules in the starch form in this part of the alfalfa plant [20].