Full Length Research Paper
ABSTRACT
The variation of some growth, agronomical and biochemical parameters of Vigna unguiculata (L. Walp) under salinity stress were investigated in the greenhouse and farm conditions. Plants were subjected to 0, 50, 100 and 200 mM NaCl. The increases of NaCl in the culture medium significantly (P< 0.001) decreased the growth parameters (number of leaves, noose diameter, leaf area and stem height), dry biomass (roots, shoots and total), chlorophyll contents and micronutrients (Zn, Fe and Mn) from 100 mM NaCl. Macronutrients (K, Ca and Mg) and K/Na ratio significantly (P < 0.001) decreased in plant partitioning with increasing salinity. The metabolites (proline, total soluble carbohydrates, soluble proteins, total free amino acids, total phenolic and flavonoids contents) significantly (P < 0.001) increased from 50 mM NaCl in variety Ekomcalle compared to Mouala GG and could be considered as biochemical indicators of early selection and osmotic adjustment ability for salt tolerant plants. At 50 mM NaCl, a relative tolerance of variety Ekomcalle compared to Mouala GG for all agronomic parameters (the flowering time, number of flowers per plant, number of pods/plant, number of seeds/pod, pod yield, seed yield and harvest index) was observed suggesting that Ekomcalle could increase cowpeas production on salty soils.
Key words: Vigna unguiculata, salinity, growth, metabolites, mineral uptake, agronomic parameters.
INTRODUCTION
In the coastal, arid and semi-arid regions, the high soil salinity severely impacts crop production by limiting plant growth and development (Ashraf and Ali, 2008; Santhi et al., 2013; Gouveitcha et al., 2021). In the other words, salinity negatively influences the homeostatic balance of water potential and ion uptake within a plant. Under high salinity, sodium toxicity may cause a range of disorders affecting germination, development, photosynthesis, protein synthesis, lipid metabolism, leaf chlorosis, and senescence (Rahneshana et al., 2018). The responses to the changes carried out by salt stress are often accompanied by a decrease in leaf area, increase of leaf thickness and succulence, abscission of leaves, necrosis of plant, and decrease of internode lengths (Parida and Das, 2005; Rahneshana et al., 2018). Salinization of soil is caused by two main factors: high evaporation relative to precipitation in association with weak leaching in soils, and salt accumulation as a result of the use of saline water (Singh, 2015; Hand et al., 2021). Previous workers like Rhoades and Loveday (1990), Santhi et al. (2013), and Hand et al. (2017) estimated that, nowadays 20% of the world’s cultivated land and nearly half of all irrigated lands are affected by salinity.
Facing the detrimental effects of salinity stress in many species, compatible osmoprotectants, such as proline, total soluble carbohydrates, proteins content and total free amino acids are produced to protect the cells against the adverse effects from salt stress (Meguekam et al., 2014; Nouck et al., 2016; Hand et al., 2021). Plants develop several mechanisms that induce tolerance to overcome salinity effects like the osmotic adjustment mechanism which consists of the sequestration of large amounts of salt ions in the vacuole and/or synthesis of organic osmolytes (Munns, 2002; Rais et al., 2013). The impact of salinity stress and mineral nutrient solution on growth and productivity has been largely studies on different crop plants (Li et al., 2008; Taffouo et al., 2010; Hand et al., 2021). It has been observed that high levels of sodium (Na) inhibit potassium (K), calcium (Ca) and magnesium (Mg) uptake in leaves, which results in a K/Na antagonism. Net photosynthesis is affected strongly by NaCl conditions as it is related directly to the closure of stomata due to low levels of intercellular CO2 (Turan et al., 2007). Plants cultivated in saline areas often showed microelements such as zinc (Zn), ion (Fe) and manganese (Mn) deficiency symptoms due to their low solubility (Hand et al., 2021). NaCl may change the solubility of these micronutrients and their available concentration in the soil (Sharply et al., 1992).
Cowpea is an economically important crop with nutritional and medicinal values. It can consist of 25% protein and is low in anti-nutritional factors, provides a rich source of proteins and calories, as well as minerals and vitamins (Sreerama et al., 2012). Their cultivation enables farmers and traders to generate more income, provide employment opportunities and so improve the economy of the nation. With the rising trend of global population, the demand for food is also on the increase. Cowpeas are grown for dry seeds and as leafy vegetables in different parts of the world (Summerfield et al., 1974; Singh et al., 2003). They resist drought stress and can recover rapidly during vegetative growth stage by re-watering because of their efficiency in using soil water (French, 1998). The main challenge in salty areas therefore is how to enhance plant growth to improve crop production under saline conditions. In order to address these challenges to the world’s food security, the identification of salt tolerant crop is considered as a valuable tool to enhance productivity and develop sustainable agriculture in salt affected lands. The main objective of this work was to compare the responses of two cowpeas cultivars exhibiting differences in salt tolerance on the growth, chlorophyll content, macro and micronutrient uptake, metabolites and agronomic parameters under saline stress in order to identify a potential biochemical indicator for early selection of salt-tolerant plants and discuss the physiological responses and adaptative strategies.
MATERIALS AND METHODS
Study area and plant
The study was carried out in a greenhouse of Faculty of Science, The University of Bamenda located in Bambili-Cameroon, with elevation of 1444 m, found in Mezam division of the NorthWest region of Cameroon. The work was carried out from December 2020 to April 2022. Average rain fall, and temperatures are 854 mm/year and 30°C and relative humidity is nearest to 84%. Prevailing winds carry the tropical monsoon. The seeds of two varieties of cowpeas (Ekomcalle and Mouala GG) used for the experiment were obtained from the breeding program of Agronomic Institute Research and Development (IRAD) Nkolbisson, Yaounde-Cameroon. The two varieties were chosen for their resistance to pathogens and socio-economic rank.
Plant growth conditions and salt treatments
Cowpeas seeds were sterilized after a viability test with 3% of sodium hypochlorite for 10 min, washed eight times with demineralized water and planted into 2 L polythene bags previously filled with 2 kg of sterilized sand, with one plant each and five replications per treatment. The plants were arranged in a complete randomized block design and daily supplied with a modified nutrient solution (in g L-1): of 150 g Ca(NO3)2, 70 g KNO3, 15 g Fe–EDTA, 0.14 g KH2PO4, 1.60 g K2SO4, 11 g MgSO4, 2.5 g CaSO4, 1.18 g MnSO4, 0.16 g ZnSO4, 3.10 g H3BO4, 0.17 g CuSO4 and 0.08 g MOO3 (Hoagland and Arnon, 1950). The pH of the nutrient solution was adjusted to 7.0 by adding HNO3 0.1 mM. Plants were subjected to different salt concentrations (0, 50, 100 and 200 mM NaCl) with 0 mM NaCl as a control in the culture medium for a period of six weeks to determine the physiological and biochemical responses of cultivars to salt stress. The average day and night temperatures in the greenhouse were between 25 and 18°C, respectively during the growth period with average relative air humidity of 75%. Parameters evaluated under greenhouse conditions: number of leaves, noose diameter, stem height, leaf area, dry biomass (roots and shoots) and ratio (roots/shoots), chlorophyll (a+b), total soluble proteins, total free amino acids, proline, total soluble carbohydrates, total phenol and flavonoids content and mineral (Na, K, Ca, Mg, Fe, Mn and Zn contents) of roots and shoots.
Plant growth
The leaf area, stem height, number of leaves, noose diameter, and dry weights were recorded after six weeks. The leaf area was calculated using the formula, surface area (cm2) = 1/3 (length × width). The parts (Roots and shoots) of the plant were dried separately (roots and shoots) at 65°C for 72 h in an oven and their dry biomasses were determined (Ndouma et al., 2020). The stem height was determined by measuring with a ruler.
Mineral uptake
In order to extract Na, K, Ca, Mg, Fe, Zn and Mn, 2 g of dried plant organs were separately added to 20 mL of Hcl for 24 h. The filtrate was analysed with an atomic absorption spectrophotometer (Rayleigh WFX-100) using the Pauwels et al. (1992) method.
Chlorophyll content
The Arnon (1949) method was used to determine chlorophyll (a+b) content. 1 g of fresh cowpeas leaves were crushed, and their contents extracted with 80% of alkaline acetone (v/v). The filtrate was analyzed using a spectrophotometer (Pharmaspec model UV-1700) at 645 and 663 nm wavelengths.
Biochemical parameters
Primary metabolites
Soluble protein content: PR content was determined by Bradford (1976) method. An appropriate volume (0 - 100 µl) of sample was put into a test tube and the total volume was augmented to 100 µl with distilled water. 1 mL of Bradford working solution was added to the sample. Then the mixture was thoroughly mixed with a vortex mixer. The absorbance
was read at 595 nm with a spectrophotometer UV (PG instruments T60) after 2 min. The standard curve was used to determine PR content.
Proline content: PRO was estimated using Bates et al. (1973) method. 0.5 g of fresh leaves were weighed, crushed and put inside a flask. 10 mL of 3% aqueous sulphosalicylic acid was poured in the same flask. The mixture was homogenized, and then filtered with a Whatman No. 1 filter paper. 2 mL of filtered solution was poured into a test tube, and then 2 mL of glacial acetic acid and ninhydrin acid were respectively added into the same tube. The test tube was heated in a warm water bath for 1 h. The reaction was stopped by placing the test tube in an ice bath. 4 mL of toluene was added to the test tube and stirred. A purple-coloured mixture was obtained and its absorbance was read at 520 nm by spectrophotometer UV (Pharmaspec model UV-1700). The concentration of PRO was determined using the standard curve (µg/g FW).
Soluble carbohydrate content: CH was obtained using phenol-sulphuric acid (Dubois et al., 1956). The fresh leaves (1 g) were ground in 5 mL of 80% ethanol and filtered with the Whatman No. 1 filter paper. The extract was diluted with deionized water to make up 50 mL. 1 mL of sample was poured in test tube, followed by the addition of 1 mL of phenol solution and 5 mL of sulphuric acid. The mixture was then swirled. The absorbance was read at 490 nm using a spectrophotometer (Pharmaspec UV-1700 model). The quantity of CH was deduced from the glucose standard curve.
Total free amino acids content: FAA content was determined by the ninhydrin method (Yemm and Cocking, 1955). Fresh leaves (1 g) were ground in 5 mL of ethanol 80%, amino acids were then extracted using reflux technique in boiling ethanol for 30 min. After decanting, the supernatant was filtered using Whatman No. 1 filter paper. The filtrate was collected, and the residue used to repeat the extraction. The two filtrates were mixed, and the raw extract of amino acid content was measured using ninhydrine method. The absorbance of purplish-blue complex was read at 570 nm wavelength. The standard curve was established using 0.1 mg/mL of glycine.
Secondary metabolites
Flavonoids content: FLA of crude extract was determined by using the aluminium chloride colorimetric method (Chang et al., 2002). 50 µL of crude extract (1 mg/mL ethanol) was made up to 1 mL with methanol, mixed with 4 mL of distilled water and then 0.3 mL of 5% NaNO2 solution; 0.3 mL of 10% AlCl3 solution was added after 5 min of incubation, and the mixture was allowed to stand for 6 min. Then, 2 mL of 1 mol/L NaOH solution was added, and the final volume of the mixture was brought to 10 mL with double-distilled water. The mixture was allowed to stand for 15 min, and absorbance was recorded on spectrophotometer (Pharmaspec UV-1700 model) at 510 nm wavelength. FLA content was calculated from a grutin calibration curve, and the result was expressed as grutin equivalent per g dry weight.
Total phenolic content: TP content of the extract was determined by the Folin Ciocalteu method (Marigo, 1973). Subsamples (1 g) of fresh leaves were ground at 4°C in 3 mL of 0.1 N HCl. After incubation at 4°C for 20 min, the homogenate was centrifuged at 6000 g for 40 min. The supernatant was collected, the pellet re-suspended in 3 mL of 0.1 N HCl and centrifuged as previously. The two supernatants were mixed. 15 µL of the mixture was extracted and 100 µL Folin-Ciocalteu reagents, 0.5 mL of 20% Na2CO3 added before incubating at 40°C for 20 min and. the absorbance was read at 720 nm using a spectrophotometer (Pharmaspec UV-1700 model). A standard curve was established using chlorogenic acid. TP content was expressed as mg g-1 fresh weight.
Agronomic parameters
The farm experiment was done at the University of Bamenda agricultural research farm located in Bambili-Cameroon. Bambili, elevation 1444 m, found in Mezam division of the NorthWest region of Cameroon. The work was carried out from March 2020 to April 2022. Average rain fall, and temperatures are 854 mm/year and 31°C and relative humidity is nearest to 84%. Prevailing winds carry the tropical monsoon. Table 1 shows the physico-chemical properties of the soil taken from 0 to 20 cm depth of the experimental site in Bambili. The plots were arranged in a randomized complete block design within a split-plot layout with two main treatments 0 and 50 mM NaCl, three replications and 0 as a control. The surface area of the plots was 5×1 m surface with intra spacing of 1.5 m. The cultivars were planted at 0.50 m spacing. Data from crop yield were collected from fifteen plants per repetition for each variant of the experiment. The agronomic parameters assessed were the flowering time, number of flowers per plant, number of pods/plant, number of seeds/pod, pod yield, seed yield and harvest index. The number of flowers was determined by counting flowers every week for each treatment until the emergence of the first pods. The number of pods per plant was determined every week for each treatment until harvest time. The flowering time was gotten by noting the date of first appearance of flower for each treatment. The yield was obtained:
Yield (t/h) = Total production (tonne)/surface (hectare).
The harvest index (HI) was calculated (Bijalwan and Manmohan, 2014):
HI = WP/(WP+ Biomass (shoot and root)) × 100 [30]
where HI = Harvest Index and WP = Weight of pods.
Statistical analysis
The experiment was performed using a completely randomized design. All data were presented in terms of mean (± standard deviation), statistically analysed using Graph pad Prism version 5.01 and subjected to analysis of variance (ANOVA). Statistical differences between treatment means were established using the Fisher Least Significant Difference (LSD) at P < 0.05. Probability level was calculated using Duncan’s Multiple Range Test (DMRT).
RESULTS AND DISCUSSION
Plant growth
Growth parameters
The inhibition effect of salinity on the growth of V. unguiculata was significantly (P<0.01) noted from 100 mM NaCl for both study varieties (Table 2). The cultivar Ekomcalle showed greater tolerance to NaCl treatment than cultivar Mouala GG in all growth parameters. The interactions cultivar × salinity was significant (P<0.05) for stem height and leaf area total dry weight (Table 1). These results are in line with the findings of Rais et al. (2013), Hand et al. (2017), and Ndouma et al. (2020). These authors explained that salinity stress negatively affects plant growth and photosynthetic functions of crop plants due to the presence of excessive amounts of Na+ and Cl- ions which cause osmotic effects, nutrients imbalance, and inhibition of enzymes activities, cell division and affect plants metabolism.
The number of leaves decreased significantly (p < 0.05) with increased salt concentration from (100 mM NaCl) (Table 2). These results are consistent with the reports of Shrivastava and Kumar (2015) on soil salinity and Kamran et al. (2020) on hazardous impacts on soil salinity. These authors reported that soil salinity leads to a reduction in the amount of water taken up by plants and the accumulation of Na+ in the leaves, eventually injuring the cells. The noose diameter significantly (p < 0.05) reduced with increasing salinity (100 mM NaCl) (Table 2). These results are substantiated by those of Nassar et al. (2020) on Triticum aestivum L. and Santhi et al. (2013) on Solanum nigrum L. According to their findings, the reduction of noose diameter with increasing salinity was due to physiological responses from the plant due to modifications of the ionic balance, mineral nutrition and photosynthetic efficiency which affect the formation of both phloem and cells. Similar observations were made for leaf area and stem height. Both parameters also decreased with increasing soil salinity. A significant decrease (p < 0.05) of leaf area and stem height was observed from 100 mM NaCl (Table 2). Heidari et al. (2014) affirmed this pattern in his work, stating that the loss of water by plant cells caused the loss of cell turgor and shrinkage, reducing the rate of cell elongation. This contributed to the formation of shorter plant stems and smaller leaves with reduced leaf area. Karam et al. (2020) in conformity with this trend reported that photosynthetic rates were retarded under high salinity due to decreased efficiency of growth hormones, resulting in decreased stem height and low water uptake, leading to lesser leaf area development. In this study, the improvement observed in the Ekomcalle variety was much more for all growth parameters as compared to the Mouala GG variety. This is because, effect of salinity on photosynthetic activities, ions imbalance, cell division and growth of Ekomcalle crop plant are less than in the Mouala GG variety (Rais et al., 2013; Heidari et al., 2014).
Dry weight partitioning
Generally, salinity has a negative impact on plant dry biomass. The dry biomass of V. unguiculata significantly decreased (p < 0.001) in plant partitions with increased salinity (Table 2). These results are in conformity with those of Heidari (2012). He explained that the reduced ability of plants to take in water under saline conditions caused slower growth and also that excessive salts from the transpiration stream injured the cells of transpiring leaves and finally contribute to reduced plant biomass. The results also showed that the dry biomass of the roots decreased significantly (p < 0.01) from 100 mM NaCl for variety Mouala GG while those of the Ekomcalle variety decreased significantly (p < 0.05) at the same concentration. The same observations were seen in the shoots and total dry biomass of both varieties. The dry biomass of variety Ekomcalle improved more compared to variety Mouala GG in all concentrations and plant parts (Table 2). This can be explained by the inhibition of minerals uptake in the tissues and decreased in the photosynthetic activities of Mouala GG variety which caused the decrease in dry biomass as compared to that of the Ekomcalle variety (Hand et al., 2017; Ndouma et al., 2020). It was also observed that, the reduction in water uptake and the hydrolysis of food reserves from storage tissue to the developing embryo (Menguekam et al., 2014) were less for the Ekomcalle variety. This is proof that this variety is more tolerant compared to Mouala GG. The ratio root/shoot dry biomass decreases significantly (p < 0.05) in the culture medium from 100 mM NaCl for both varieties (Table 2). These results corroborate those of Assimakoupoulou et al. (2015) on three green bean cultivars. He explained that the decreased of root/shoot ratio may improve salinity tolerance by restricting the flux of toxic ions to the shoot, delaying the onset of the tolerance threshold.
Mineral distribution
The Na+ concentration significantly increased (p < 0.001) with increased salinity in both shoots and roots while K+, Ca2+ and Mg2+ decreased significantly (p < 0.001) (Table 3). According to Assaha et al. (2017), Na+ in plants increased with increased salinity due to imbalance in the Na+/K+ transporters. Similarly, Greenway and Munns (1980) and Taffouo et al. (2010) showed that, the ratio is distorted by the high salt concentration in the soil, which enhanced the uptake of Na+ and Cl- and affects the uptake of other minerals. Previous authors like Munns and Tester (2008) and Rais et al. (2013) explained that the competition of Na+ and K+ for aerial plants resulted in greater accumulation of Na+ in the shoots than in the roots. It can be also caused by the loss of osmotic potential of root medium, specific ion toxicity and the lack of nutritional ions. In the same line, Marschner (1995) and Rahneshan et al. (2018) showed that K+ plays a crucial role in the regulation of stomata movement, activation of the enzymes needed in the metabolism, proteins and carbohydrates synthesis, osmotic adjustment and cell turgor.
In this study, Ekomcalle variety accumulated more Na+ in the shoots and roots compared to Mouala GG variety and higher accumulation is observed in the shoots compared to the roots in both varieties. This can be explained by the fact that Ekomcalle variety easily translocates the Na+ from the roots to the shoots causing the leaves to get dry and fall rapidly. This can be considered as a tolerant mechanism for the tolerant glycophytes under salinity.
Ca2+ decreased significantly (p < 0.001) in the culture medium with increased salinity (Table 3). According to Rahneshan et al. (2018), Ca2+ ameliorates salt stress through osmotic adjustments by enhancing the selective absorption of potassium and other minerals in plants (Schachtman and Liu, 1999). In saline medium, Hosseni and Thengane (2007) and Hand et al. (2017) also indicated that the uptake of Na+ inhibits the uptake and transportation of other mineral elements to the leaves. Na+ replaces Ca2+ resulting in the disintegration of cell membranes and cell walls and acts as a secondary messenger in the regulation of signal transduction pathways for the response to abiotic stress and in the promotion of K+/Na+ selectivity (Shabala et al., 2006). In the present work, the decrease of calcium from membranes and cell walls by sodium of Ekomcalle variety can be suggested to be one of the responses to salinity stress tolerance compared to Mouala GG variety.
The decrease of Mg2+ with increased salinity as reported by Shabala et al. (2006) and Hand et al. (2017) was attributed to the Na+/K+ antagonism which favoured the uptake of Na+ over other minerals (Table 3). Rahneshan et al. (2018) also reported that many key chloroplast enzymes are strongly influenced by slight variations in levels in the cytosol and chloroplast. Our results revealed a decreased Mg2+ content in both cultivars partitioning. This decrease had less influence Ekomcalle and Mouala GG growth due to Mg2+ deficiency, and the activity of some enzymes, which requires Mg2+ for catalysis as well as chlorophyll synthesis (Khan et al., 2000; Rahneshan et al., 2018).
The increased intake doses of NaCl in the culture medium generally influenced the Fe, Mn and Zn. These minerals decreased significantly (p < 0.05) in plant partitioning (Table 3). This can be explained by the mineral disorder, the competition for mineral uptake, transport or mineral distribution within the crop plant (Munns and Tester, 2008). The presence of Nacl in the soil leads to change and increased the solubility of study micronutrients and their availabilities (Sharply et al., 1992). In this work, Fe, Mn and Zn contents were higher in the shoots as compared to the roots for both varieties. These results are in consonance with those of Marschner (1995). He explained that the ability of genotypes of plants to metabolize micromineral vary under salinity stress and depend on the plant crop. The mineral contents were higher in Ekomcalle variety compared to Mouala GG in all concentrations and plant parts. We can say that the variety Ekomcalle is more tolerant than Mouala GG.
Chlorophyll (a+b) content
In general, salinity has detrimental effects on chlorophyll (a+b). The effect of increased soil salinity was observed at 100 mM NaCl, with a significantly reduction (p < 0.05) in Mouala GG as compared to Ekomcalle at the same concentration (Figure 1). These results are in consonance with those of Santhi et al. (2013) on S. nigrum; Najar et al. (2019) on Medicago truncatula. They explained that salinity contributes to stomatal closure which reduces CO2 assimilation in the leaves and conduct to the reduction of photosynthetic activities. In other words, the accumulation of salts in older leaves leads them to eventually die and drop off. In this study, the photoinhibitory damages caused by chlorophyllase or reactive oxygen species (ROS) formation under salt stress explained by Heidari et al. (2014) and Najar et al. (2019) had less effect on variety Ekomcalle as compared to Mouala GG.
Metabolites
Primary metabolites
The increased concentrations of NaCl in the culture medium significantly increased (p < 0.001) the total free amino acids, total soluble carbohydrates, soluble proteins and proline in all cultivars (Figure 2). These results are in consonance with the findings of Hasegawa et al. (2000), Munns and Tester (2008), Chelli-Chaabouni et al. (2010) and Gouveitcha et al. (2021). They said that metabolites like FAA, CH, PR and PRO are salt-tolerance indicators and possess osmoprotective qualities which are one of the common responses of plants to changes in the external osmotic potential. In this work, the PRO content of variety Ekomcalle and Mouala GG increased with the intake doses of salt from 50 mM NaCl. The PRO accumulation was much more marked in variety Ekomcalle compared to Mouala GG. This can be explained that Ekomcalle variety is more tolerant than Mouala GG. It is considered to be compatible regulator due to its accumulation under salinity stress. According to previous workers, Solomon et al. (1994), Smirnoff and Cumbes (1989), Venekamp (1989) and Gouveitcha et al. (2021), PRO plays the role of protective and stabilizing agent for enzymes and membranes; a free radical scavenger, a carbon and nitrogen storage compound and the regulation of cytosolic pH, respectively.
The total free amino acids increased with increased intake doses of NaCl during the experimentation (Figure 2). The findings of Cusido et al. (1987), Ndouma et al. (2020), Hand et al. (2017) and Negrao et al. (2017) showed that salinity increased the levels of total free amino-acids due to the reduction of osmotic potential to maintain the turgid potential and may be a good indicator for screening salt-tolerant genotypes. These results are in consonance with the findings of Kosová et al. (2013) and Hand et al. (2017). They reported that soluble proteins increased due to regulatory adjustments to stress resulting in its active synthesis. This is because soluble proteins enhance plant salt tolerance. Its production is considered as an adaptive mechanism of plants to salinity.
From the results obtained, the total soluble carbohydrates increased significantly with salinity in the leaves of two cowpeas varieties. According to Movafegh et al. (2012), the accumulation of total soluble carbohydrates in plant tissues under salinity conditions stress was due to regulatory osmotic adjustment in current stress. The osmotic balance of the cytoplasm relies on an active synthesis of organic compounds such as soluble carbohydrates which enhances the plant salt tolerance through osmotic adjustment (Hajiboland et al., 2014).
Secondary metabolites
The results of non-enzymatic antioxidants (total phenolic (TP) and flavonoid (FLA)) accumulated under stress condition as shown in Figure 2. This is corroborated by the findings of Menguekam et al. (2014) and Ndouma et al. (2020). These authors maintain that, the accumulation of TP and FLA are physiological responses to plant stress. Their accumulation is a cellular adaptive mechanism for scavenging oxygen free radicals, while maintaining chlorophyll levels and cell turgor to protect photosynthetic activities. These results agree with those of Radi et al. (2013) who reported that salinity stress affects the phenolic compounds content by the induced disturbance of the metabolic processes leading to an increase in phenolic compounds. This study showed that an increase in salt concentrations led to a significant increase in total phenol contents. According to Hichem et al. (2009), the high accumulation of phenolics compound in plants physiologically is important in overcoming the salinity-induced oxidative stress.
Agronomic parameters
The yield components were generally influenced by salinity in the field (Table 4). The number of flowers per plant, number of pods/plant, number of seeds/pod, pod yield, seed yield and harvest index were significantly (p < 0.05) decreased in the presence of NaCl for both varieties (Table 4). According to Alam et al. (2004), salinity might have reduced the production of crop by overturning water and nutritional balance of plant and loss of photosynthetic capacity. The latter is a limiting factor to the supply of carbohydrates for plant grow. Our findings showed that the variety Ekomcalle improves all agronomic parameters compared to Mouala GG variety. Munns (2002) explained that salinity reduced plant parts development by reducing turgor in growing plant roots and shoots due to limited water potential in roots growth medium, consequently Ekomcalle is more tolerant than Mouala GG. In addition, Villora et al. (2000) showed that the yield of tolerant crops could not be affected by a low level of salinity, even though the leaf area and the shoot biomass are reduced.
CONCLUSION
The conducted study confirmed that the two cowpeas varieties were generally affected by NaCl stress throughout the entire experiment. The results revealed that, the growth parameters (number of leaves, stem height, leaf area and noose diameter), the dry biomass (roots, shoots, total and roots/shoots), the mineral uptake (K, Ca, Mg, Zn, Fe Mn and K/Na) and Chlorophyll (a+b) decreased significantly with increasing intake doses of NaCl from 100 mM NaCl while Na+ and metabolites (PRO, PR, CH, FAA, TP and FLA) increased from 50 mM NaCl. In the field experiment, agronomic parameters (flowering time, number of flowers per plant, number of pods/plant, number of seeds/pod, pod yield, seed yield and harvest index) were improved at 50 mM NaCl in the variety Ekomcalle compared to Mouala GG at the same concentration. The variety Ekomcalle was less affected by the detrimental impacts of salinity resulting to significant improvements in all the studied parameters compared to variety Mouala GG. The high accumulation of metabolites with increased salinity doses, the dry biomass and chlorophyll content could be added as indicators of early identification and of osmotic adjustment ability for salt-tolerant plants in salt stress conditions. The varieties Ekomcalle and Mouala GG could be cultivated in areas with moderate salinity with preference given to the Ekomcalle variety.
COMPETING INTERESTS
The authors have declared any conflict of interests.
REFERENCES
|
Alam MZ, Stuchbury T, Naylor REL, Rashid MA (2004). Effect of salinity on growth of some modern rice cultivars. Joural of Agronomy. 3:1-10. |
|
|
Ashraf M, Ali Q (2008). Relative membrane permeability and activities of some antioxidant enzymes as the key determinants of salt tolerance in canola (Brassica napus L.). Environmental and experimental Botany 63(1-3):266-673. |
|
|
Assaha DVM, Ueda A, Saneoka H, Al-Yahyai R, Yaish MW (2017). The Role of Na+ and K+ Transporters in Salt Stress Adaptation in Glycophytes. Frontiers in Physiology 8:509. |
|
|
Assimakopoulou A, Salmas I, Nifakos K, Kalogeropoulos P (2015). Effect of salt stress on three green bean (Phaseolus vulgaris L.) cultivars. Notulae Botanicae Horti Agrobotanici Cluj-napoka 43(1):113-118. |
|
|
Arnon DI (1949). Copper enzymes in isolated chloroplasts. Polyphenylodase in Beta vulgaris. Plant Physiology 24(1):1-15. |
|
|
Bates L, Waldren RP, Teare ID (1973). Rapid determination of free proline for water- stress studies. Plant and Soil 39(2):205-207. |
|
|
Bijalwan A, Manmohan JRD (2014). Productivity of Wheat (Triticum aestivum) as Intercro pin Grewia Optiva Based Traditional Agroforestry System along Altitudinal Gradient and Aspectin MidHills of Garhwal Himalaya, India. American Journal of Environmental Protection 5(2):89-94. |
|
|
Bradford MM (1976). A rapid and sensitive method for quantitation of microgram of protein utilizing the principle of protein- dye binding. Anal Biochemistry 72(1-2):248-254. |
|
|
Chang CC, Yang MH, Wen HM, Chern JC (2002). Estimation of Total Flavonoid Content in Propolis by Two Complementary Colorimetric Methods. Journal of Food Drug Analysis 10(3):178-182. |
|
|
Chelli-Chaabouni A, Ben Mosbah A, Maalej M, Gargouri K, Gargouri-Bouzid R, Drira N (2010). In vitro salinity tolerance of two pistachio rootstocks: Pistacia vera L. and P. atlantica Desf. Environmental and Experimental Botany 69(3):302-312. |
|
|
Cusido RM, Palazon J, Altobella T, Morales C (1987). Effect of salinity on soluble protein, free amino acids and nicotine contents in Nicotiona rustica L. Plant Soil 102:55-60. |
|
|
Dubois M, Gilles KA, Hamil JK, Rebers PA, Smith F (1956). Colorimetric method for determination of sugars and related substances. Anal Chemistry 28(3):350-356. |
|
|
French RJ (1998). Effects of early water deficit on growth and development of faba bean plant. Plant Physiology 116:447-53. |
|
|
Greenway H, Munns R (1980). Mechanism of salt tolerance in nonhalophytes. Annual Reviews of Plant Physiology 31:149-190. |
|
|
Gouveitcha MBG, Kpinkoun JK, Mensah ACG, Gandonou CB (2021). Salinity resistance strategy of okra (Abelmoschus esculentus L. Moench) cultivars produced in Benin Republic. International Journal of Plant Physiology and Biochemistry 13(1):19-29. |
|
|
Hajiboland R, Norouzi F, Poschenrieder C (2014). Growth, physiological, biochemical and ionic responses of pistachio seedlings to mild and high salinity. Trees 28:1065-1078. |
|
|
Hand MJ, Taffouo VD, Nouck AE, Nyemene KPJ, Tonfack LB, Meguekam TL, Youmbi E. (2017). Effects of Salt Stress on Plant Growth, Nutrient Partitioning, Chlorophyll Content, Leaf Relative Water Content, Accumulation of Osmolytes and Antioxidant Compounds in Pepper (Capsicum annuum L.) Cultivars. Notulae Botanicae Horti Agrobotanici Cluj-Napoca 45(2):481-490. |
|
|
Hand MJ, Nono GV, Tonfack LB, Taffouo VD, Youmbi E (2021) Nutrient Composition, Antioxidant Components and Ascorbic Acid Content Response of Pepper Fruit (Capsicum annuum L.) Cultivars Grown under Salt Stress. International Journal of Biology 2(1):43-70. |
|
|
Hasegawa PM, Bressan RA, Zhu J-K, Bohnert HJ (2000). Plant cellular and molecular responses to high salinity. Annual Review of Plant Physiology and Plant Molecular Biology 51:463-499. |
|
|
Heidari M (2012). Effects of salinity stress on growth, chlorophyll content and osmotic components of two basil (Ocimum basilicumL.) genotypes. African Journal of Biotechnology 11(12):379-384. |
|
|
Heidari A, Bandehagh A, Toorchi M (2014). Effects of NaCl Stress on Chlorophyll Content and Chlorophyll Fluorescence in Sunflower (Helianthus annus L.) Lines. Yuzuncu Yil University Journal of Agricultural Sciences 24(2):111-120. |
|
|
Hichem H, Mounir D, Naceur EA (2009). Differential responses of two maize (Zea mays L.) varieties to salt stress: Changes on polyphenols composition of foliage and oxidative damages. Industrial Crops Production 30 (1):144-151. |
|
|
Hoagland DR, Arnon DI (1950). The water-culture method for growing plants without soil. University of California, College of Agriculture, Berkley pp. 1-34. |
|
|
Hosseni G, Thengane RJ (2007). Salinity tolerance in cotton (Gossypium hirsutum L.) genotypes. International Journal of Botany 3(1):48-55. |
|
|
Kamran M, Parveen A, Ahmar S, Malik Z, Hussain S, Chattha MS, Saleem MH, Adil M, Heidari P, Chen J-T (2020). An Overview of Hazardous Impacts of Soil Salinity in Crops, Tolerance Mechanisms, and Amelioration through Selenium Supplementation. International Journal of Molecular Sciences 21(1):148. |
|
|
Khan MA, Ungar IA, Showlter AM (2000). The effect of salinity on growth, water status, and ion content of a leaf succulent perennial halophyte, Suaeda fruticosa (L.) Forssk. Journal of Arid Environments 45:72-85. |
|
|
Kosová K, Prášil IT, Vítámvás P (2013). Protein Contribution to Plant Salinity Response and Tolerance Acquisition. International Journal of Molecular Sciences 14:6757-6789. |
|
|
Li N. Chen, Zhou S, Li X, Shao C, Wang J, Fritz R, Hüttemannn E, Polle A (2008). Effect of NaCl on photosynthesis, salt accumulation, and compartmentation in two mangrove species, Kandelia candel and Bruguiera gymnorrhiza. Aquatic Botany 88:303-310. |
|
|
Marigo G (1973). On a fractionation method and estimation of the phenolic compounds in plant. Analysis 2(2):106-110. |
|
|
Meguekam TL, Taffouo VD, Grigore MN, Zamfirache MM, Youmbi E, Amougou A (2014). Differential responses of growth, chlorophyll content, lipid perodxidation and accumulation of compatible solutes to salt stress in peanut (Arachis hypogaea L.) cultivars. African Journal of Biotechnology 13(50):4577-4587. |
|
|
Movafegh S, Roghie RJ, Shadi K (2012). Effect of salinity stress on chlorophyll content, proline, water soluble carbohydrate, germination, growth and dry weight of three seedling barley (Hordeum vulgare L.) cultivars. Journal of Stress Physiology and Biochemistry 8(4):157-168. |
|
|
Munns R (2002). Comparative physiology of salt and water stress. Plant Cell Environment 25:239-250. |
|
|
Munns R, Tester M (2008). Mechanisms of salinity tolerance. Annual Review of Plant Physiology 59:661-681. |
|
|
Najar R, Aydi S, Sassi-Aydi S, Zarai A, Abdelly C (2019). Effects of salt stress on photosynthesis and chlorophyll fluorescence in Medicago truncatula. Plant Biosystems 153(1):88-97. |
|
|
Nassar RMA, Kamel HA, Ghoniem AE, Alarcón JJ, Sekara A, Ulrichs C, Abdelhamid MT (2020). Physiological and Anatomical Mechanisms in Wheat to Cope with Salt Stress Induced by Seawater. Plants (Basel) 9(2):237. |
|
|
Ndouma MC, Nouck AE, Titah MA, Ndjouondo GP, Ekwel SS, Fotso, Taffouo VD (2020). Growth parameters, mineral distribution, chlorophyll content, biochemical constituents and non-enzymatic antioxidant compounds of white yam (Dioscorea rotundata (L) var. gana) grown under salinity stress. GSC Biological and Pharmaceutical Sciences 12(03):139-149. |
|
|
Negrao S, Schmockel SM, Tester M. (2017). Evaluating physiological responses of plants to salinity stress. Annals of Botany 119(1):1-11. |
|
|
Nouck AE, Taffouo VD, Tsoata E, Dibong SD, Nguemezi ST, Gouado I, Youmbi E (2016). Growth, Biochemical Constituents, Micronutrient Uptake and Yield Response of Six Tomato (Lycopersicum esculentum L.). Journal of Agronomy 15 (2):58-67. |
|
|
Parida AK, Das AB (2005). Salt tolerance and salinity effects on plant: a review. Ecotoxicology and Environmental Safety, 60:324-349. |
|
|
Radi AA, Farghaly FA, Hamada AM (2013). Physiological and biochemical responses of salt-tolerant and salt-sensitive wheat and bean cultivars to salinity. Journal of Biology and Earth Sciences 3(1):72-88. |
|
|
Rais L, Masood A, Inam A, Khan N (2013). Sulfur and Nitrogen Co-ordinately Improve Photosynthetic Efficiency, Growth and Proline Accumulation in Two Cultivars of Mustard Under Salt Stress. Journal of Plant Biochemistry and Physiology 1(1):101. |
|
|
Rahneshan Z, Nasibia F, Moghadam AA (2018). Effects of salinity stress on some growth, physiological, biochemical parameters and nutrients in two pistachio (Pistacia vera L.) rootstocks. Journal of plant interactions 13(1):73-82. |
|
|
Rhoades JD, Loveday J (1990). Salinity in irrigated agriculture. In American Society of Civil Engineers, Irrigation of Agricultural Crops. American Society of Agronomists 30:1089-1142. |
|
|
Santhi MM, Gurulakshmi GS, Rajathi S (2013). Effect of Salt stress on Physiological and Biochemical Characteristics in Solanum nigrum L. International Journal of Scientific Research 4(3):2319-7064. |
|
|
Schachtman D, Liu W (1999). Molecular pieces to the puzzle of the interaction between potassium and sodium uptake in plants. Trends Plant Sciences 4:281-287. |
|
|
Shabala S, Demidchik V, Shabala L, Cuin TA, Smith SJ, Miller AJ, Davies JM, Newman IA (2006). Extracellular Ca2+ ameliorates NaCl-induced K+ loss from Arabidopsis root and leaf cells by controlling plasma membrane K+ permeable channels. Plant Physiology 141(4):1653-1665. |
|
|
Sharply AH, Meisinger JJ, Power JF, Suarez DL (1992). Roots extraction of nutrients associated with long-term soil management. In: Advances in soil Science, Stewart BA and Lal R. (Eds). Springer, Newyork, USA. ISBN-13: 9780387976570, pp. 151-217. |
|
|
Shrivastava P, Kumar R (2015). Soil salinity: A serious environmental issue and plant growth promoting bacteria as one of the tools for its alleviation. Saudi Journal of Biological Sciences 22(2):123-131. |
|
|
Singh A (2015). Soil salinization and waterlogging: a threat to environment and agricultural sustainability. Ecological Indicators 57:128-130. |
|
|
Singh BB, Ajeigbea HA, Tarawali SA, Fernandez-Rivera S, Musa. (2003). Improving the production and utilization of cowpea as food and fodder. Field Crops Research 84(1-2):169-177. |
|
|
Smirnoff N, Cumbes QJ (1989). Hydroxyl radical scavenging activity of compatible solutes. Phytochemistry 28(4):1057-1060. |
|
|
Solomon A, Beer S, Waisel Y, Jones GP, Paley LG (1994). Effects of NaCl on the carboxylating activity of rubisco from Tamarix jordanis in the presence and absence of proline-related compatible solutes. Physiologia Plantarum 90(1):198-204. |
|
|
Sreerama YN, Sashikala VB, Vishwas MP, Vasudeva S (2012). Nutrients and antinutrients in cowpea and horse gram flours in comparison to chickpea flour: Evaluation of their flour functionality. Food Chemistry 131(2):462-468. |
|
|
Summerfield R, Huxley PA, Steele W (1974). Cowpea [Vigna unguiculata (L) Walp]. Field Crop Abstracts 27:301-12. |
|
|
Taffouo VD, Nouck AE, Dibong SD Amougou A (2010). Effects of salinity stress on seedlings growth, mineral nutrients and total chlorophyll of some tomato Lycopersicum esculentum L. cultivars. African Journal of Biotechnology 9(33):5366-5372. |
|
|
Turan MA, Turkmen N, Taban N (2007). Effect of NaCl on stomacal resistance and proline, chlorophyll, Na, Cl and K concentrations of Lentil plants. Journal of Agronomy 6(2):378-381. |
|
|
Venekamp JH (1989). Regulation of cytosol acidity in plants under conditions of drought. Physiologia Plantarum 76(1):112-117. |
|
|
Villora G, Moreno DA, Pulgar G, Romero L (2000). Yield improvement in Zucchini under salt stress: Determining micronutrient balance. Scientia Horticulturae 86:175-183. |
|
|
Yemm EW, Cocking EC (1955). The determination of amino acids with ninhydrin. The Analyst 80:209-213. |
|
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