**2.1. Hydrolysis of storage seed proteins**

seed stores a food reserve mainly as proteins, lipids and carbohydrates [2]. Protein and oil bodies are the major reserve in oilseed which represent a source for each of energy, carbon, and nitrogen during seedling establishment [3]. Because the physiology of reserve mobilization during germination and post-germination events is still poorly understood, extensive studies must be performed to know the metabolic mechanisms of reserve food mobilization providing insights into the ability to use such seeds as planting material [4]. Enzymatic hydrolysis of protein, lipid and carbohydrate, and transportation of metabolites is dependent

Physiological and biochemical changes followed by morphological changes during germination are strongly related to seedling survival rate and vegetative growth which affect yield and quality. Food reserve of starch and protein are mainly stored in the endosperm. In general, germination process can be distinguished into three phases: phase I, rapid water imbibition by seed; phase II, reactivation of metabolism; and phase III, radicle protrusion [6]. The most critical phase is phase II whereas, the essence physiological and biochemical processes such as hydrolysis, macromolecules biosynthesis, respiration, subcellular structures, and cell

Water imbibition by reserve substances in germinating wheat seed stimulates the embryo to produce phytohormones mainly gibberellic acid (GA) which can diffuse to aleurone layer and initiate a signaling cascade resulting in the synthesis of α-amylases and other hydrolytic enzymes. Then, hydrolytic enzymes secrete into the endosperm and hydrolyzed food reserve [8, 9]. Germination is considered a response includes bidirectional interactions between the embryo and endosperm since the endosperm can secrete signals to control embryo growth [10]. Previous studies were investigated the activity of some key enzymes in glycolysis, pentose phosphate pathway (PPP), the tricarboxylic acid cycle (TCA cycle), and amino acid

Seed germination is particularly vulnerable to environmental stress encountered conditions, specifically salt and water which are widespread problem around the world [12]. High salt and drought tolerance seeds might be showed rapid germination resulting in a good seedling establishment and hence expected to maintain high yield productivity [13]. Water and salt stress conditions affect seed germination with reducing germination rate and delay in the initiation of germination [14]. Under water stress, enzymes activity such as α-amylase in *Cicer arietinum* cotyledons [15] or α- and β-amylase in *Medicago sativa* germinating seeds [16] were reduced. In contrast, water stress conditions led to an increase in the activity of α-amylase in *Hordeum vulgare* seedlings [17], β-amylase in *Cucumis sativus* cotyledons [18], cytosolic glyceraldehyde-3-phosphate dehydrogenase in *Craterostigma plantagineum* plants [19] and protease in *Oryza sativa* seedlings [20]. Salt stress causes ion toxicity, osmotic stress and reactive oxygen species (ROS) stress [21]. ROS reacts with cell macromolecules [22] and lipids [23], and disrupt diverse physiological and biochemical processes, such as hormonal imbalance and reduced use of reserves [24]. Plants develop ROS-scavenging mechanisms include enzymatic and non-enzymatic antioxidant systems [25] that protect plants against oxidative damage. Therefore, improvement the activity of antioxidant enzymes in plants organs is necessary for increasing plant's salt tolerance. Species and varieties/cultivars varied in their ability for

elongation are reactivated resulting in initiation of germination [7].

mainly on water availability [5].

142 Advances in Seed Biology

metabolism during germination [11].

Proteolytic enzymes have the main role in using stored protein in metabolism of germinating seeds which proceed through many stages [36]. According to Gepstin and Ilan [37], proteolytic activity in germinating beans increased during the first 7 days which partially dependent on the embryonic axis. Proteases and peptidases have been detected in many seeds during germination whereas; plant protease and amylase inhibitors which are proteinaceous in nature are being disappeared [38]. Antitryptic and antichymotryptic activities were observed to be markedly reduced in the endosperm of finger millet on germination which might be attributed to the proteolytic activity in hydrolysis of the inhibitory proteins [39]. Hydrolysis of stored proteins produced free amino acids, which support protein synthesis in endosperm and embryo and so proceeding of germination process [40]. Schlereth et al. [41] recorded an initial little decrease in free amino acids at the beginning of vetch seeds imbibition which is attributed to leakage from the axis, but remain without change during late germination stage.

A disulfide proteome technique was developed by Yano et al. [42] to visualize redox changes in proteins. This technique was used to analyze rice bran resulting in identification of embryospecific protein 2 (ESP2), dienelactone hydrolase, putative globulin, and globulin-1S-like protein as putative target of thioredoxin, which support the hypothesis that thioredoxin activates cysteine protease with a concurrent unfolding of its substrate during germination [43].

In buckwheat seeds, the main storage protein constituent about 16% of total seed protein is the 13S globulin with molecular mass of about 300 kDa and consists of acid and basic subunits with molecular masses ranging from 57.5 to 23.5 kDa [44]. During seed germination, 13S globulin is hydrolyzed by proteolytic enzymes through stages and the products are used by the growing seedling. The first stage of the 13S globulin degradation resulted from a limited proteolysis activity of metalloproteinase with the cleavage of about 1.5% of peptide bonds. This stage proceeds during the first 3 days of germination. It takes place during the first 3 days of germination [45]. Metalloproteinase activity is controlled by a proteinaceous inhibitor (Mr—10 kDa), present in dry buckwheat seeds in a complex with the enzyme which dissociated by bivalent cations liberated from phytin hydrolysis process. Phytin is present in buckwheat seeds in sufficient amount in the form of globoids disposed in protein bodies [46].

During the second stage of 13S globulin degradation; the products of metalloproteinase protein activity hydrolyzed into small peptides and amino acids at acid pH (5.6) by cysteine proteinase and carboxypeptidase which appear in germinating seeds [47]. It was clear that cysteine proteinase is able to hydrolyze only the modified l3S globulin but not the native. The role of carboxypeptidase is to facilitate the flow of storage protein hydrolysis and works in cooperation with cysteine proteinase. At latest stage when pH becomes more acidic (5.0) in the vacuoles, aspartic proteinase which is present in dry seeds is involved into the course of hydrolysis protein bodies.

#### **2.2. Hydrolysis of storage seed starch**

Carbohydrates represent the most storage food constituent in cereal grains, whereas it contains about 70–80% starch, about 15% protein, less than 5% lipids, minerals and vitamins. In cereals, most hydrolysis enzymes are produced in the aleurone or scutellum in response to germination signals. Several modified seed systems were used to detect the induction process and identify potential factors controlling enzyme induction in absence of the embryo [48].

Chrispeels and Varner [49] observed that isolated aleurone failed to synthesize α-amylase in a manner quantitatively similar to distal half seeds led to correction by adding calcium to the medium. The role of calcium might be expected to involve amylase stability, and to have a much more complex involvement in regulating enzyme activities [50]. Because of *de novo* amylase synthesis during seed germination to stimulate the stored starch mobilization for providing young plant till photosynthesis will be initiate, amylase has been showed high activity [51]. Parys et al. [52] showed that the amylase activity is regulated by the concentration of reducing sugars in vivo in both cotyledons and axis. At the time, the amylase activity in the cotyledons increased gradually and reached a maximum on the 5th day of germination process, while the starch decreased and soluble sugars increased [53].

Many studies which concerned with studying the essentiality of α-amylase activity during seed germination under drought stress and could be summarized as follows; the promotion of drought stressed germinating seeds is a result of high α-amylase activity directly but, it might be related to adaptive strategy to water deficit since its activity is required for solutes accumulation and decrease osmotic potential [54, 55]. In addition, α-amylase synthesis inhibition might be not a mechanism by which drought prevents the germination of *Agropyron desertorum* seeds [54]. GAs can alleviate the drought stress-caused inhibition of seed germination through regulation of α-amylase [19].
