**2.3. Hydrolysis of storage seed lipids**

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

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

hydrolysis protein bodies.

144 Advances in Seed Biology

**2.2. Hydrolysis of storage seed starch**

Generally oilseeds composed of two parts, the kernel which is main part and the seed covering that enclosed the kernel and called the husk or tegument. The kernel comprised two parts which are the embryo and the endosperm. Lipase activity is investigated during seed germination where it is maximum value [56, 57]. Triacylglycerols is stored in oleosomes and comprise in range from 20 to 50% of dry. As germination proceeds, triacylglycerols are hydrolyzed to produce energy which required for the synthesis of sugars, amino acids (mainly asparagine, aspartate, glutamine and glutamate) and carbon chains required for embryonic growth [58].

Lipid level and lipase activity were studied in various germinating seeds. It was showed that β-oxidation takes place 4 days after germination of Castor been seeds [59]. The major hydrolytic enzymes concerned with the lipid metabolism during germination are the lipases which catalyze the hydrolysis of ester carboxylate bonds and releasing fatty acids and organic alcohols [60, 61] and the reverse reaction (esterification) or even various transesterification reactions [62]. The ability of lipases to catalyze these reactions with great efficiency, stability and versatility makes these enzymes highly attractive from a commercial point of view.

Villeneuve [63] and others classified lipases specificities into three main groups; the 1st group is **substrate specificity** in which glycerol esters represent the natural substrates, the 2nd group is called **regioselective** and involves the subgroups *non-specific lipases* that hydrolyze the triacylglycerols into fatty acids and glycerol in a random way with production of monoand diacylglycerols as intermediate products (**Figure 1**); *specific 1.3 lipases* which catalyze the hydrolysis at C<sup>1</sup> and C<sup>3</sup> glycerol bonds in triacylglycerols with liberating of fatty acids and unstable intermediates 2-monoacylglycerols and 1.2-or 2.3-diacylglycerols and *specific or selective type fatty acid* that hydrolyze the ester bond of a specific fatty acid or a specific group of fatty acids at any position of triacylglycerol. The 3rd group **enantioselective** could identify enantiomers in a racemic mixture. The enantio specificities of lipases depend on the type of substrate [64].

**Figure 1.** Regioselective: non-specific and 1,3 specific lipases catalyze the hydrolysis of triglycerides in different manners with the production of fatty acids.

The induction of lipase activity during germination might be dependent on factors from embryo [65]. Early study of Shoshi and Reevers [66] showed the presence of two lipases in the endosperm of Castor been seed, acid lipase in dry seed and alkaline lipase during germination. On the other hand, storage tissues of all the oilseeds except Castor bean contained only lipase activity which increased during germination [67].

Because of sucrose is the substrate for lipid biosynthesis in developing seed and the end product of lipid degradation, it might be primarily considered as regulatory factor in studying the mechanisms of lipid metabolism [58, 68]. In addition, asparagine and nitrate are considered regulatory factors in lipid metabolism of lupine [69]. In lupin germinating seeds, the level of asparagine can reach 30% of dry matter, and it is a main transport form of nitrogen from source to sink tissues [70]. Borek et al. [71] reported that asparagine controls the metabolism of carbohydrate as it caused a significant decrease in soluble sugars and increase in starch in organs of germinating lupin seed. In contrast, nitrate is not a favorable source of nitrogen in protein metabolism in lupin seeds [72] and rather does not influence the carbohydrate metabolism [71]. Nitrate similarly as N sucrose, is regarded as a factor which can regulate plant metabolism by changes in the expression of some genes [73].

Storage lipid mobilization in germinating seeds begins with hydrolysis of triacylglycerols in oleosomes by lipases into free fatty acids and glycerol. Then fatty acids undergo β-oxidation in peroxisomes. Next, glyoxylate cycle will proceed partially in the peroxisome and partially in the cytoplasm. Three of the five enzymes of the glyoxylate cycle (citrate synthase, isocitrate lyase and malate synthase) are located in peroxisomes, while two other enzymes (aconitase and malate dehydrogenase) operate in the cytoplasm [74]. Succinate transported from peroxisome to mitochondria and here is converted to malate via the Krebs cycle. Malate in turn, after transport to the cytoplasm, is converted to oxaloacetate. Finally, gluconeogenesis and the synthesis of sugars are the processes which are a form of carbon transport especially in germinating seeds proceed [58, 75].

#### **2.4. Hydrolysis of phytic acid during seed germination**

The induction of lipase activity during germination might be dependent on factors from embryo [65]. Early study of Shoshi and Reevers [66] showed the presence of two lipases in the endosperm of Castor been seed, acid lipase in dry seed and alkaline lipase during germination. On the other hand, storage tissues of all the oilseeds except Castor bean contained only

**Figure 1.** Regioselective: non-specific and 1,3 specific lipases catalyze the hydrolysis of triglycerides in different manners

Because of sucrose is the substrate for lipid biosynthesis in developing seed and the end product of lipid degradation, it might be primarily considered as regulatory factor in studying the mechanisms of lipid metabolism [58, 68]. In addition, asparagine and nitrate are considered regulatory factors in lipid metabolism of lupine [69]. In lupin germinating seeds, the level of asparagine can reach 30% of dry matter, and it is a main transport form of nitrogen from source to sink tissues [70]. Borek et al. [71] reported that asparagine controls the metabolism of carbohydrate as it caused a significant decrease in soluble sugars and increase in starch in organs of germinating lupin seed. In contrast, nitrate is not a favorable source of nitrogen in protein metabolism in lupin seeds [72] and rather does not influence the carbohydrate metabolism [71]. Nitrate similarly as N sucrose, is regarded as a factor which can regulate

Storage lipid mobilization in germinating seeds begins with hydrolysis of triacylglycerols in oleosomes by lipases into free fatty acids and glycerol. Then fatty acids undergo β-oxidation in peroxisomes. Next, glyoxylate cycle will proceed partially in the peroxisome and partially in the cytoplasm. Three of the five enzymes of the glyoxylate cycle (citrate synthase, isocitrate lyase and malate synthase) are located in peroxisomes, while two other enzymes (aconitase and malate dehydrogenase) operate in the cytoplasm [74]. Succinate transported from peroxisome to mitochondria and here is converted to malate via the Krebs cycle. Malate in turn, after transport to the cytoplasm, is converted to oxaloacetate. Finally, gluconeogenesis and the synthesis of sugars are the processes which are a form of carbon transport especially in

lipase activity which increased during germination [67].

with the production of fatty acids.

146 Advances in Seed Biology

plant metabolism by changes in the expression of some genes [73].

germinating seeds proceed [58, 75].

The greatest storage form of total phosphorus (about 50–80%) is phytic acid (C<sup>6</sup> H18O24P6 ) and also known as inositol hexophosphate (IP6) in legumes and cereals seeds [76]. Phytic is regarded as antinutrient because it has the ability to form complexes with proteins and bind with cations (especially Fe, Ca, K, Mn, Mg, Zn) via ionic association to form a mixed salt called phytin or phytate with the reduction of their digestive availability [77]. On the other hand, phytate may play an important role as an antioxidant by forming iron complex that cause a decrease in free radical generation and the peroxidation of membranes, and may also act as an anticarcinogen, providing protection against colon cancer [78]. Because of it was regarded as antioxidant, anticarcinogen or vitamin like substance, it is essential to measure and manipulate phytate content in food grains such as beans [79, 80].

One of the major breeding objectives is the development of crop cultivars with low seed phytin content. It was found that the increase in *myo-*inositol and reduced amounts of *myo*inositol phosphate intermediates in the seeds of maize mutants with a phenotype of reduced phytic acid had a little effect on plant growth and development [81]. These findings might suggest that a high level of stored phytate is not necessary for seed viability and germination or seedlings growth.

Phytin is mainly stored in protein bodies in seeds called globoids in the aleurone layer and scutellum cells of most grains. Phytic acid has a strong ability to chelate multivalent metal ions, specially zinc, calcium, iron and as with protein residue. Seed phytate content depend mainly on the environmental mainly plant phosphorus fertilization [82]. It has been shown the important genetic variability in the phytate content of beans and it appears to be a trait controlled by several genes [83]. Also, a correlation between phytate and protein contents was found [84], so the protein content of grains can be considered another factor that regulates phytate content.

Phytin in germinating seeds is hydrolyzed by an acid phosphatase enzyme called phytase [85], with releasing of phosphate, cations, and inositol which are utilized by the seedlings. It was found little changes in extractible P<sup>i</sup> in hazel seeds during chilling accompanied with IP6 mobilization that might be suggested the rapid conversion of Pi into organic form [86]. These results were discussed as evidence of active metabolism in germinating seed [87]. In agreement, phytase is strongly and competitively inhibited by Pi , while the decrease in phytase activity coincided with maximal IP6 turnover [88]. It was found that about 87% of IP6 is digested during the first 6 days of germination [89]. In this respect, Ogawa et al. [90] postulated that the early axiferous IP6 digestion is essential for metabolic activity of the resting tissue via supplying Pi and minerals for physiological and metabolic requirements, for example, enzymes of starch metabolism. In addition, IP6 related compounds such as pyrophosphatecontaining inositol phosphates (PP-IP) play a potential role in providing Pi for ATP synthesis during the early stages of germination before complete dependence on aerobic mitochondrial respiration the mainly source of ATP production [91].

In stressed seeds, many vital processes such as germination, growth, respiration and other related processes are affected which consequently can trigger other effects on metabolic activities particularly the enzymes of phosphate metabolism that play an important role in germination and seed development [92]. Phosphate metabolism is one of negatively affected processes under different stressful conditions [93]. Under stressful conditions, the restriction of growth and phosphorus availability resulting in enhancement the activity of phosphatases to produce Pi by hydrolysis the insoluble phosphate form that modulate mechanism of free phosphate uptake. In agreement, Olmos and Hellin [94] reported that acid phosphatases activity increased to sustain Pi level which enables it to be co-transported with H+ down a proton motive force gradient.
