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. Comparing with adult plant, the mechanisms of stress tolerance in germinating phase are poorly interpreted and might be related to a series of factors that are inherent to the species and environment [ 26 , 27 ].
Phytohormones are considered the most important endogenous substances for modulating physiological and molecular responses [ 28 ]. The strigolactone SL are relatively new phytohormones. Genetically and physiological studies have been demonstrated the effective roles of the plant hormones ABA and GAs in regulation of dormancy and germination [ 29 ].
To counteract the adverse effects of abiotic stress, seed priming methods have been applied to improve germination, uniformity, improve seedling establishment and stimulate vegetative growth in more field crops [ 30 , 31 ]. Wheat seeds were priming to increase germination characteristics and stress tolerance.
As seeds imbibe water, metabolic processes initiate with an increase in respiration rate [ 7 ]. Early developmental stages of seedling require fueling energy before it becomes autotrophic [ 32 ]. Seeds store mineral nutrients as sucrose or amino acids which are synthesized into starch or proteins during development to be used in early seedling emergence.
The role of hydrolytic enzymes in seed germination On seed hydration, separate intercellular bodies of seed stored carbohydrates, proteins, lipid and phosphate act as energy source and carbon skeleton [ 33 ]. Seed imbibition triggered many metabolic processes such as activation or freshly synthesis of hydrolytic enzymes which resulted in hydrolysis of stored starch, lipid, protein hemicellulose, polyphosphates and other storage materials into simple available form for embryo uptake.
Hydrolysis of storage seed proteins Proteolytic enzymes have the main role in using stored protein in metabolism of germinating seeds which proceed through many stages [ 36 ]. 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 ].
This technique was used to analyze rice bran resulting in identification of embryo-specific 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 ]. 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. 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. 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.
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 ].
The role of calcium might be expected to involve amylase stability, and to have a much more complex involvement in regulating enzyme activities [ 50 ]. 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 ].
Hydrolysis of storage seed lipids 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 ]. 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. 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. The 3rd group enantioselective could identify enantiomers in a racemic mixture. The enantio specificities of lipases depend on the type of substrate [ 64 ].
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 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.
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 ]. 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. Credit: Chathurika Wijewardana Seed depth On your package of seeds, there are instructions for how deep to plant your seeds.
This is another area where optimal depth will depend on the plant. Small seeds typically need to lay on top of the soil for successful germination. Because of their small size, they only have stored food for a limited period of growth. If we put small seeds in too deep, lack of oxygen will limit seed germination, or the seedling will finish its food reserve prior to reaching the soil surface. On the other hand, large seeds need a deep planting location so that roots can grow deeply for proper anchorage.
In your home garden, when you start planting your seeds vegetables, flowers, or any kind of herbs , the two most important things that you want to achieve are maximum germination and fast germination rate.
However, due to some natural factors and environmental limitations you may have to put an extra effort to achieve the best possible results. These are some tips you can use to achieve the highest germination rate: Always plant seeds that are for that particular year. If you feel tempted to use seeds purchased in a previous year, do a germination test first see bottom of linked page. Choose the ideal planting time, as noted on your seed packets.
The ideal planting temperature range will be listed. Some seeds stay dormant and take a long time to germinate until they have enough moisture to grow. Pre-soaking the seeds before planting usually overnight, on a wet paper towel , can help.
By doing so, you can protect your plant from any damages like wind, frost, or drought and later you can move the seedlings outdoors to continue to develop. Overall, the diversity and proteolytic specificity of barley proteases involved in the degradation of storage compounds makes them a starting point to select a potential candidate for further applications. Potential Application of Barley Proteases in Celiac Disease From an applied point of view, this wide knowledge on barley proteases acting in the germination process could be employed to manipulate and improve the grain composition not only in barley but also in those cereal species such as wheat, oat, rye and related species and hybrids, in which a number of proteins, known as gluten proteins, affect the health of sensitive consumers.
Celiac disease is the best-characterized pathology associated with gluten consumption and there is a major environmental factor, the ingestion of gluten proteins not only from wheat but also from barley and rye. A lifelong gluten free diet reverses signs and symptoms of celiac disease and NCWS. Nevertheless, this is difficult to follow due to the wide gluten presence in many diet foods. Most of the CD related epitopes have been found in the gliadin fraction Arentz-Hansen et al.
Gliadins are rich in the amino acids proline, and glutamine, which make them resistant to being fully digested in the gastrointestinal tract. This is the case of human proteases, which do not accept proline at their cleavage sites.
According to this model, a first innate immune response is triggered by certain peptides, such as the mer gliadin peptide, resulting in the production of interleukin 15 IL by epithelial cells. The result is the disruption of the epithelial barrier by increasing its permeability. Different therapeutic alternatives are being developed, as the inhibition of transglutaminase, the antagonism of peptide binding to HLA-DQ2 or HLA-DQ8, the enzymatic detoxification of gluten, or the introduction of natural amino acid substitution to eliminate toxicity Schuppan et al.
Some other strategies have been developed trying to relieve the negative effect of gluten proteins Scherf et al. A promising approach is the down-regulation of genes encoding for gliadins by RNAi technologies, generating transgenic wheat lines with low levels of toxicity for celiacs Gil-Humanes et al.
Previous investigations have already developed wheat with a down regulation of gliadins expression by hairpin technology. As result, a very low or null T-cells stimulation was proven Gil-Humanes et al. Their results showed that targeting of genes related to celiac disease is feasible and may reduce T-cell epitopes.
With the use of these four CysProt as a representation of different groups of L-like cathepsins and F-like cathepsins, a wide view of their activity on gliadin proteolysis has been achieved. The final goal has been to explore their potential to reduce gliadin content in the wheat gluten or to use them in therapy treatments. Hydrolysis of the different wheat gliadins by purified barley CysProt was observed by western blot analysis.
When gliadins from the six wheat cultivars were treated with HvPap-1, -4, -6, and for 1 and 12 h, different degradation patterns by each CysProt were observed Figure 2A.
Degradation in all gliadin samples was appreciated after 1 h of incubation with HvPap-1, -4, -6, and proteases. In most cases, higher bands remained stable while lower molecular weight bands started to disappear after 1 h of treatment. After 12 h of incubation with barley proteases, an increased differential degradation of gliadin bands was observed.
To discard any processing or degradation effect due to instability or autohydrolysis, gliadins were incubated for 12 h without proteases Figure 2B. Likewise, as HvPap-1, -4 and were activated by adding pepsin, an analysis of gliadins stability in presence of this commercial peptidase was carried out Figure 2B.
Although some smaller bands were hydrolysed by pepsin, in all treatments the band with higher molecular weight remains stable after 12 h incubation, in contrast with the total degradation exerted by HvPap-4 and HvPap These proteases seem to be the most promising gliadin-degrading enzymes.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 ]. Most seeds will not germinate under waterlogged conditions, because water is taking up all the air space in the soil. It was showed that the reactivity of dehydrogenases covered the first 3 days of cowpea seed germinations [ ]. Although these lotus offspring are phenotypically abnormal, the viability of old seeds was evidently not affected by accumulated doses of up to 3 Gy. Nitrate similarly as N sucrose, is regarded as a factor which can regulate plant metabolism by changes in the expression of some genes [ 73 ]. During seed germination, 13S globulin is hydrolyzed by proteolytic enzymes through stages and the products are used by the growing seedling.
Degradation in all gliadin samples was appreciated after 1 h of incubation with HvPap-1, -4, -6, and proteases. Early developmental stages of seedling require fueling energy before it becomes autotrophic [ 32 ]. 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 ]. Moisture Moisture essentially brings the seed back to life. In conclusion, this is an alternative approach for enzymatic gluten degradation to generate gluten-free wheat to manufacture gluten free products.
Highly leakage of cellular solutes due to initial imbibition indicates cellular membranes damage caused by rehydration. The cellular mechanism of priming as it relates to improved stress tolerance in germinating seeds is still required more study.
Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. This is the case of the HvLeg-2 legumain of barley, which is highly expressed during germination and could be involved in the mobilization of storage proteins either by direct proteolytic degradation or by processing and activation of other CysProts Cambra et al.
This technique was used to analyze rice bran resulting in identification of embryo-specific 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 ]. A variety of cellular processes in plants are under control of phytohormones which play key roles and coordinate various signal transduction pathways during abiotic-stress response [ ]. The priming techniques improve stress acclimation mechanisms during germination but the cellular mechanism of priming is still requires more studying. HvPap-6 CysProt ranks as the best candidate given the encouraging results of this study. Alongside to glyoxylate cycle, the OPPP operates where a number of enzymes and intermediates participate the two pathways [ ].
Lipid level and lipase activity were studied in various germinating seeds. These are some tips you can use to achieve the highest germination rate: Always plant seeds that are for that particular year.
Plants have developed unique strategies including a tight regulation of germination ensuring species survival [ 95 ]. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. So, suitable moisture is needed to get the best results. The induction of lipase activity during germination might be dependent on factors from embryo [ 65 ]. The interpretation of suggestion that import pathway capacity is absolutely dependent on the presence of oxygen aerobic respiration is related to the significant decrease in capacity of the general import pathway in mitochondria under anaerobic conditions, compared to under aerobic conditions.
Schematic representation of Hordeum vulgare grain germination. To sustain a good seedling development, seed stores a food reserve mainly as proteins, lipids and carbohydrates [ 2 ]. Gliadins are rich in the amino acids proline, and glutamine, which make them resistant to being fully digested in the gastrointestinal tract.