Biochemical Investigations on Vigour Enhancement in Fresh and Aged Seeds Upon Seed Priming in Cowpea [Vigna unguiculata (L.) Walp.]

Seed quality is one the key factors affecting successful farming, but this trait inevitably decline in their ability to germinate and emerge into vigorous seedlings, leading to problems for successful crop production (Powell et al., 2000). Seed deterioration due to ageing is a natural and inexorable phenomenon which is regulated by various metabolic activities especially related to protein and lipid metabolism as well as the generation of free radicals and antioxidant system present in the seed. Seed ageing is also the major cause of reduced seed quality in vegetable species, leading to slow and asynchronous germination. An approach to their seed improvement has been the development of seed invigoration treatments based on seed hydration. The major cause of a decline in the vigour of vegetable seeds is believed to be seed ageing, a hypothesis that is supported by the ability of vigour tests based on ageing, such as controlled deterioration, to identify vigour differences (Matthews, 1980). The apparent cell death was attributed to rapid water uptake into the outer cells of the cotyledons, leading to disruption of cell membranes and resulting in high solute leakage from the embryos, reduced respiration and germination and a decline in both the transfer of food reserves from the cotyledons to the growing axis and the growth rate of the surviving seedlings (Powell and Matthews, 1978). Damage to the organization of cell membranes during seed ageing is an important factor in explaining seed deterioration. Accelerated ageing resulted in increased lipid peroxidation, decreased levels of antioxidants and reduced activity of several enzymes involved in scavenging of free radicals and peroxide (Khan et al., 2016). Repair mechanism of DNA is an important constituent of the “pregerminative metabolism,” which is activated with the start of seed imbibition, accompanied by uncontrolled build up of reactive oxygen species (ROS) (Paparella et al., 2015). Pre-germination treatments represent the physiological methods that improve plant production by modulating the metabolic activities of germination before the emergence of the radicle. It was suggested that reactive oxygen species (ROS) play a crucial role in signalling seed germination (Lilya Boucellia et al., 2019).
        
Cowpea seeds are more sensitive to storage conditions like high temperature; high seed moisture content, light exposure and an extended storage period have been found to adversely affect quality. Damage to the organization of cell membranes during seed ageing is an important factor in explaining seed deterioration. Pre-soaking the seeds in osmotic solutions has been demonstrated to improve the viability and vigour of aged seeds in various horticultural crops (Bhanuprakash et al., 2010). Seed priming of fresh and accelerated cowpea seeds increased the electrical conductivity of seed leachate, cell membrane stability, total protein content, a-amylase activity, peroxidise activity and dehydrogenase activity over fresh and aged dry seeds (Arun et al., 2017). Siri et al., (2013) reported that osmopriming (PEG-6000 with -1.5 MPa for 6 days) of artificially aged seeds of sweet pepper (42°C and 100% RH) resulted in an improved germination with decreased levels of malondialdehyde (MDA) and total peroxide concentration. They further stated that accumulation of total antioxidant activity (TAA), total ascorbate, dehydroascobate and catalase (CAT) activity in primed seeds enhanced the defense mechanism in protecting the cell membrane damage from reactive oxygen species. Clissia Barboza da Silva and Julio Marcos-Filho (2019) reported that priming with 24-EpiBL promoted positive effects on germination speed, seed tolerance to heat stress and initial development of seedlings with responses to in lower vigour seed in bell pepper. Transcriptomic and proteomic studies have revealed up regulation or down regulation of a number of genes and proteins during seed priming (Kubala, 2015). Advanced germination metabolism, increased activity of antioxidants, decreased level of malondialdehyde, altered ratio of saturated and unsaturated fatty acids and increased level of hydrolyzing enzymes are some of the changes associated with seed priming (Yacoubi et al., 2011). Priming could enhance the activity of antioxidative systems, resulting in lower rate of lipid peroxidation, contributing to seed invigoration and allows some of the metabolic process for germination to occur (Bradford, 1990). Priming increases the production of antioxidant enzymes like catalase, peroxidase, superoxide dismutase etc that help in protecting the cell against membrane damage because of lipid peroxidation. Very little known about the basis of vigour enhancement in aged cowpea seeds upon seed priming through biochemical investigations. With this endeavour the present investigation in this direction was chosen in order to understand how seed priming mitigate the ageing effects in cowpea. The objective of this study was to increase activity of antioxidant enzymes and protein banding pattern in cowpea with seed priming treatments.

The data pertaining to seed moisture as influenced by the vigour levels and hydro priming and chemical priming in cowpea are presented in the Table 1. The seed moisture differed significantly due to vigour levels with different chemical and hydro priming and their interaction. Among vigour levels, higher seed moisture (11.24%) was recorded in high vigour seeds and lower seed moisture (10.78%) in low vigour seeds. Among the inorganic salts and hydro priming treatments higher seed moisture (11.42%) was recorded in P₃ which was statistically on par with P₁ (11.25%), P₂ (11.17%) and P₅ (11.10%) but it was lowest (10.27%) in control (P₈).
 



        
The final germination count exhibited significant variations among vigour levels, different seed priming and their interaction between V × P. Among the vigour levels, final germination count was higher (95.66%) in V₁ and it was lower (62.33%) in V₂. Among the chemicals, higher (84.35%) at final count was registered in P₁ but it was on par with P₃ (84.0%) and P₂ (81.67%). It was the lowest (71.0%) in control (P8). Among V × P interaction, higher (98.7%) final germination count was registered in V₁P₃ and it was statistically on par with V₁P₁ (98.0%), V₁ P₄ (97.31%) and V₁P₂ (96.72%). Lowest germination in final count was recorded in V₂P₈ (50.7%) that is low vigour seed which was unprimed. Among the low vigour seeds, highest (70.71%) germination was recorded in V₂P₁ which was on par with V₂P₃ (69.38%). Good quality of seed is the basic input for success in vegetable production programme. However, age-induced seed deterioration of vegetable crops is an inexorable phenomenon which gets in the way of successful vegetable production. Audi and Mukhtar (2009) revealed that seeds treated with GA₃ hormone in cowpea significantly increased the germination percentage which may be due to increased in vigour, increased peroxide scavenging enzymatic activities and decreased lipid peroxidation. Sedghi et al., (2011) indicated that the loss of seed vigour can cause the reduction in germination percentage due to chromosomal aberrations and increase in lipid peroxidation, which may cause cell damage and seed death. As such seed deterioration caused by ageing and its repair during early germination determine the success or failure of vegetable production system. Seed deterioration can be defined as the loss of quality, viability and vigour either due to ageing or effect of adverse environmental factors. While ageing may be considered as progressive decline in biological functions accompanied by an increased risk of degenerative changes and death over time. The rate of deterioration rapidly increases with increase in seed moisture content, storage duration or temperature of storage. Loss of seed viability following ageing has been attributed to a series of metabolic defects that accumulate in embryonic and non-embryonic structures. At the cellular level, seed ageing is associated with various alterations including loss of membrane integrity, solute leakage, reduced energy metabolism, impairment of RNA (protein synthesis) and DNA degradation. Seed priming treatment i.e. slowly imbibing and then re-drying of seeds accomplished by soaking of seeds in a solution of low water potential, has been shown to reinvigourate the aged seeds. The reversal of ageing effects by seed priming has been explained by reduction of malondialdehyde (MDA) and free radicals production and maintenance of antioxidant activities due to DNA repair and favourable metabolic balance.
        
Results showed alteration in the isozyme profiles in primed and unprimed seeds. In the present investigation dry seeds when compared to primed seeds exhibited more activation of enzyme and more of isoforms. Changes in esterase profile Plate 1 and Fig 1 were noticed in both fresh and accelerated aged primed seed over control. In the present investigation, when compared to dry seeds, primed seeds exhibited more activation of enzyme. Changes in esterase were noticed due to seed ageing and subsequent priming. Seed ageing (50% seed lot) has shown loss of isomorphs of esterase isozyme when compared to control. The banding profile of esterase revealed polymorphism among priming treatments with the presence of twelve bands with Rm values ranged from 0.514 to 0.941. Low intense bands (Bands 1 to 4) of Rm value ranged from 0.514 to 0.607, revealed polymorphism since they appeared only in primed treatments. Bands 7 and 8 (Rm: 0. 714 and 0.806, respectively) of varied intensity were clearly visible in all the priming treatments however, the polymorphism was revealed only with their intensity. Band number eleven with low intensity (Rm: 0.912) appeared in most of the treatments, but it was absent in both high and low vigour unprimed control. Esterase is a hydrolyzing enzyme that splits ester into acid and an alcohol in a chemical reaction with water called hydrolysis. Increased esterase activity has been reported in various types of stress affecting plants and it has been suggested as a defense mechanism against oxidative damage. Priming allowed repair system to combat sub cellular damage activated enzyme synthesis and thus restored deterioration process started due to ageing. The increase in the germination of primed seeds might be due to increase in the synthesis of these enzymes. Similar results also have been reported in various crops (McDonald, 1999). The increase in the germination of primed seeds might be due to increase in the synthesis of the enzymes. Priming allowed repair system to combat sub-cellular damage activated enzyme synthesis and thus restored deterioration process started due to ageing (Varier et al., 2010). Thornton et al., (1993) suggested that damage to DNA which accumulates during the seed ageing is repaired by aerated hydration treatments as also during early hours of germination.
 

 

        
The detailed electrophorogram of peroxidase isozyme of primed and unprimed seeds is depicted in Plate 2 and Fig 1. Several enzymes have been investigated in relation to seed priming which are associated with early hours of germination by various scientists on a variety of crops. An attempt was made to examine the role of peroxidase upon priming in fresh and accelerated ageing seed in cowpea. The banding profile of peroxidase (POX) isozyme  revealed polymorphism due to priming treatments with a total of six bands having Rm value ranging from 0.070 to 0.554 (Plate 3). Band number one, four and six (Rm value: 0.070, 0.518 and 0.554, respectively) expressed their presence with elevated intensity in all the priming treatments over accelerated unprimed control.  However polymorphism was observed only with the intensity of bands. While band four (Rm: 0.467) was not noted in high vigour seeds when primed with CaCl₂, KBr, Mg (NO₃)₂, ZnSO₄ and hydro- primed. In contrast these bands appeared in low vigour seeds. Further, it was absent in high vigour unprimed seeds, but noticed in low vigour unprimed seeds. This clearly articulate that peroxidase enzyme would express only in stress condition. The results clearly supported that priming will restore the lost seed vigour in aged seeds due to reactivation of enzyme activity. It is also recognised that peroxidase isozyme is mostly contained to root system, but in the present study since fully imbibed seeds were used as source enzyme, complete expression of the enzyme did not perceived as such enough polymorphism could not be established in peroxidase isozyme. In canola seeds it has (Aboutalebian and Nazari, 2017) been demonstrated that seed priming treatments effectively slow down the physiological deterioration under natural and accelerated aged seeds.
 

 
 
Electrophoretic separation of total soluble seed protein of primed seeds by SDS PAGE 
 
In the present study, variations in fresh and accelerated aged seeds for proteins were noticed based on either presence or absence of specific bands at specific Rm value. Seed priming restored almost entire protein profile that was lost due to seed ageing. This may be due to the reason that the protein disappeared after subjecting the seeds for ageing treatment and reappeared after priming.
        
Banding intensity and relative mobility of total soluble seed proteins (SDS-PAGE) of primed seeds of cowpea are depicted in Plate 3 and Fig 2. Total soluble see d proteins extracted from cowpea seeds were separated by SDS-PAGE  and analysed based on zymogram pattern. The entire protein profile was divided into seven regions A-H (Region A: >97.4 kDa; Region B: 75.2.0 to 97.4 kDa; Region C: 68.7 to 75.2 kDa; Region D: 43.0 to 68.7 kDa; Region E: 29.0 to 43.0 kDa; Region F: 14.3 to 29.0 kDa; Region G: 6.5 kDa to 14.3 kDa and Region H: 3.0 to 6.5 kDa), equivalent to their increasing Rm values and decreasing molecular weight. The presence or absence of specific band or group of bands as well as band intensity was taken as the criteria to differentiate the different priming treatments in cowpea. The priming treatments from 1 to 16 was scored as low, medium and high intensity bands with Rm value ranged from 0.167 to 0.940. All the priming treatments showed significant difference either by the presence or absence of bands as well as their intensity. In region A (>97.4 kDa; phosphorylase b) there were no bands in all the non-primed and priming treatments.
 

 
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In region B (75.2 to 97.4 kDa; Bovine Serum Albumin), only two low intensity bands were seen in seed priming GA₃ and ammonium molybdate in high and low vigour seeds at Rm value of 0.166 to 0.188. In Region C (43.0 to 68.7 KD; Ovalbumin) there were four bands (3, 4, 5 and 6) with Rm value ranging from 0.285 to 0.410. Band number three was present in all the priming treatments and absent in low vigour seeds. Band number four was present in all priming treatments in high vigour seeds and missing in primed seeds of low vigour seeds. Band number five of low intensity was noticed in all the treatment except in control low vigour seeds. Band number six was present in all the treatment, but with medium intensity in high and low vigour seeds primed with GA₃, ammonium molybdate and calcium chloride. Band seven of low intensity was present in fresh and accelerated seeds when primed with GA₃, ammonium molybdate and calcium chloride and missing in all other treatments. In region C (68.7 to 75.2 kDa), there were four bands with Rm value ranging from 0.285 and 0.410. Polymorphism was observed with respect to band number 4, 5 and 7. Band number four with low intensity was present only in high vigour unprimed and as well as primed seeds. Band seven were polymorphic in nature by their absence in high and low vigour seeds primed with KBr, Mg (NO₃)₂, ZnSO₄ and hydro primed and unprimed seed of low vigour seeds. However, the expression of new proteins of low and medium molecular weights in all priming treatments were related to priming induced proteins in contrast to their absence in the unprimed seeds which are necessary for vigorous, uniform germination and longevity of seeds. Gurusinghe et al., (2002) also opined that the induction of heat–shock protein (hsp70) in tomato seeds, the abundance of Bip (78kD binding protein) and ‘class I small hsp’ in primed seeds with PEG subjected to post-priming treatment could have been involved in the extension of seed longevity.
        
In this Region D (43.0 to 68.7 kDa; Carbonic Anhydrase) total five bands with Rm value ranging from 0.436 to 0.506 were exhibited. Band number eight of medium intensity bands was noticed in high vigour seed primed with GA₃ and of low intensity bands when high vigour seeds were primed with ammonium molybdate, calcium chloride, potassium bromide, magnesium nitrate, zinc nitrate and hydro priming and low vigour seed when primed with GA₃. Band number ten of low intensity was present in all the treatment except control and low and high vigour seeds primed with GA₃. However, band eleven of high intensity was present in all the priming treatment except control low vigour seeds. Band number twelve of low and medium intensity was present in all the treatment except in low vigour seeds when primed with GA₃, ammonioum molybdate, calcium chloride, potassium bromide, magnesium nitrate, zinc sulphate and hydro priming.
        
In this region E (29.0 to 43.0 KD; Trypsin Inhibitor) total of four bands with Rm value that ranged from 0.561 to 0.635. Band number thirteen with Rm value of 0.561 medium intensity bands was present only in seed primed with ammonium molybdate and low intensity bands were present in GA₃ and calcium chloride and absent in all the other treatments. Band number fourteen with Rm value of 0.584 low intensity bands was present in high vigour seeds were primed with chemicals and absence of band in low vigour seeds. Band number fifteen with Rm value 0.608 of varied intensity was present in all the priming treatment except when low vigour seeds were primed with various chemicals. However, band number sixteen with Rm value 0.635 low and medium intensity bands was present in all the priming treatments except in when low vigour seeds were primed with GA₃, ammonium molybdate, calcium chloride, potassium bromide, magnesium nitrate, zinc sulphate and hydropriming. However, in region E (20KDa) there was four bands with Rm value ranging from 0.561 to 0.635. Band number thirteen with low intensity was present in high as well as low vigour unprimed seeds and also in GA₃, ammonium molybdate and calcium chloride primed seeds. Wahid et al., (2008) reported variation in protein expression of sunflower achenes after priming with various chemicals. Priming showed little improvement in the banding pattern and intensity in normal seeds, while low-vigour seeds had significant improvement in the banding pattern and intensity of protein. Loss of desiccation tolerance in germinating seeds is associated with degradation of LEA proteins.
        
In region F (Lysozyme- 14.3 to 29.0 kDa), a total of five bands with Rm value ranging from 0.661 to 0.753 was observed. Band number seventeen with Rm value of 0.661 of low intensity was present in all the treatments except in control accelerated aged seeds. Band number eighteen with Rm value 0.677 showed low and medium intensity bands in all the treatments. Band number nineteen with Rm value 0.710 of low intensity was present in all the treatments. But, band number twenty with Rm value 0.738 of high intensity was noticed in all the priming treatment. Band number twenty one of high intensity band was noticed in all the treatment except control accelerated aged seeds. Polymorphic in nature but it was not comparable among high and low vigour seeds.
        
In the region Region G (Aprotinin- 6.5 to 14.3 kDa) there was total of four bands with Rm value ranging from 0.802 to 0.931 was observed. In band number twenty two low intensity bands were present in all the primed treatments and missing in control fresh and accelerated seeds as similar to that of twenty two. However, in band number twenty three low intensity bands were present in all the treatments. Band number twenty four of medium intensity was present in all the priming treatments except in control high vigour seeds and missing in all the low vigour cowpea seeds. In band number twenty five with Rm value 0.931 high intensity bands were noticed in all the treatments. According to Fujikura and Karssen (1992), appearance of vigour related proteins noticed during priming which might have enhanced the expression new protein (P1) at 25kD and combination of ageing and priming enhanced protein expression (Q1) at 24 kD.
        
In region H (Insulin- 3 to 14.3 kDa), only one band of low intensity with Rm value of 0.966 was appeared in all the priming treatments except in low vigour Mg (NO₃)₂, ZnSO₄, hydro primed and unprimed seeds. The disappeared proteins may be related to desiccation tolerance. However, the total soluble seed protein profile (SDS-PAGE) revealed polymorphism between the priming treatments in the regions B, C, E, G and H which also suggested that upon priming, synthesis new proteins had occurred. Thus the results showed that priming will restore the lost seed vigour in aged seeds due to reactivation of proteins in aged seeds and expression of these proteins in priming treatments are related to priming induced proteins in contrast to their absence in the aged seeds which are necessary for germination and longevity of seeds. Haroun and Hussein (2003) reported that seed priming increased the intensities of protein bands from 20-32 kDa in Lupinus. The quantitative increase in protein profile might be due to the presence of all the components necessary for resumption of protein synthesis except polysomes within the cells of mature dry embryos. However polysomes might have been formed immediately after inbibition by combination of single ribosomes which might have initiated the process of protein synthesis as suggested by Bewley (1997). The variations in the appearance of bands may be due to activation of membrane bound enzymes which actively participate in protein synthesis necessary to carry out complete emergence of seedlings. Wedad and Samha (2006) reported a unique higher intensity protein band in the GA₃ primed radish seedling and the band was absent in the unprimed seedling under stress condition. This indicated relatively higher protein concentrations in the primed seedlings than the unprimed seedlings. Abbas and Alkadious (2013) reported that the total number of protein bands in the leaves of cowpea treated with carrot roots extract and germinated under 100 mM NaCI was increased (10 bands) as being compared to the respective control (8 bands). Kubala et al., (2015), found positive association of improved germination in PEG-osmoprimed brassica seeds with protein synthesis and reported higher expression of proteins involved in water transport, modification in cell wall, cell division and management of oxidative stress after PEG-osmopriming. Thus the protein analysis study clearly revealed the appearance and disappearance of peptides at specific Rm values that can be employed as a marker for priming.
        
The total soluble seed protein profile had revealed polymorphism expression (26 bands with values ranging from 0.167 to 0.940) with respect to appearance and disappearance of peptides at specific Rm values. The present study concluded that priming in aged seeds of cowpea increased enzyme activity, restored almost entire protein profile and esterase isozyme profile as it allowed repair system to combat sub-cellular damage, activated enzyme synthesis and thus restored deterioration process to certain extent started due to ageing. Thus based on the findings, it is to be concluded that in cowpea, the loss of viability in aged seeds was due to impaired metabolism as evident from lower levels of enzyme synthesis and higher membrane injury
        
The changes in the activities of the enzyme, upon priming, suggest that mobilization of storage material may be responsible for increased germination and vigour in primed seeds when compared to unprimed aged seeds. The protein seed profile analysis can be employed as a marker for priming. Therefore priming can be used as a good technique for enhancing vigour of low vigour seed lots of cowpea.