The following points highlight the three main biochemical changes during germination. The changes are: 1. Respiration 2. Protein and Nucleic Acid Synthesis 3. Mobilization of Stored Reserves.
Biochemical Change # 1. Respiration:
In the imbibed seed, all the three respiratory pathways, viz., glycolysis, pentose phosphate pathway and citric acid cycle are active in varying degrees. These play a vital role in the production of key intermediates in metabolism, energy in the form of ATP and reducing power as NADH and NADPH.
Glycolysis, catalysed by cytoplasmic enzymes, can operate under aerobic and anaerobic conditions to produce pyruvate. In the absence of oxygen, it is reduced further to ethanol and carbon ox the presence of oxygen, pyruvate is further metabolized within the mitochondria.
Here pyruvate undergoes oxidative decarboxylation to acetyl CoA which is completely oxidized to CO2 and H2O via the citric acid cycle. ATP molecules are generated during oxidative phosphorylation when electron are transferred from the reduced coenzymes, NADH and FADH down an electron transport a though a series of electron carriers located on the inner membrane of mitochondria to modular oxygen.
The PP pathway is important because it produces NADPH, the readily available reducing power within the cytoplasm for utilization in reductive biosynthesis.
Even the dry seeds contain about 10-15 per cent water. Low levels of respiration have been reported in dry seeds. A very notable metabolic event during imbibition occurring in less than 15 min is the reformation of keto acids from amino acids by deamination and transamination.
It has been suggested that these keto acids, important for respiratory pathways, are stored in the dry seeds as the corresponding amino acids, and then reformed on hydration. Respiration can be divided into four phases.
Phase I:
This is characterized by a sharp rise in respiration for about 10 hours and is due to the activation and hydration of mitochondrial enzymes belonging to the cycle and electron transport chain.
Phase II:
This involves a lag in respiration between 10 and 25 hours after the start of imbibition. Hydration of the cotyledons is now completed and all pre-existing enzymes activated. It is interesting to note that there is rapid oxygen uptake into seeds with intact testas during phase I (early imbibition), whereas the same testa impedes oxygen uptake in phase II. Between phase II and phase III, the radicle penetrates the testa.
Phase III:
A second respiratory surge characterizes this phase which is thought to be due to increased oxygen supply through pierced testa. Another reason for respiratory increase should be the newly synthesized mitochondria and respiratory enzymes in the dividing cells of the growing axis.
Phase IV:
This is characterized by a marked fall in respiration that coincides with the disintegration of the cotyledons following exhaustion of the stored food. It has been shown that in the early stages of germination, respiration is cyanide-resistant and the alternative oxidase instead of cytochrome oxidase plays a role in germination.
Cyanide has been shown to stimulate lettuce and Amaranthus seed germination and inhibitors of alternate oxidase such as salicylhydroxamates (SHAM) and chlorbenzhydroxamates (CLAM) inhibit germination by about 50 per cent. At later stages of germination, however, respiration becomes sensitive to cyanide.
The P/O ratio of cyanide-resistant respiration is one (in case of NAD-linked substrates) and zero (in case of succinct) in contrast to three for conventional respiration. The pro-motive effect of respiratory inhibitors on germination may be due to large scale participation of pentose phosphate pathway instead of glycolysis and citric acid cycle and the PP pathway is in some way connected with initiation of germination.
Biochemical Change # 2. Protein and Nucleic Acid Synthesis:
It is generally recognized that protein synthesis is a Pre-requisite for radicle emergence Protein synthesis does not occur in the dry seeds but starts when these are hydrated and cytoplasmic ribosomes (eukaryotic SOS) get associated with messenger RNA (mRNA).
Initiation of protein synthesis in which embryo involves the attachment of the small (40S) ribosomal subunit and the initiating tRNA molecule (methionyl tRNA) to the initiation site on the mRNA.
After formation of the initiation complex the large (60S) ribosomal subunit becomes attached and protein synthesis begins. Formation of the initiation complex requires CTP and perhaps ATP.
Other soluble protein factors are required for the transfer of aminoacyl-tRNAs to their appropriate codons on the ribosome-mRNA complex, and to move the message through the ribosome. These are the elongation factors requiring CTP for activity.
One of the controversial questions regarding protein synthesis in the early imbibitional phase is whether prior RNA synthesis, especially, mRNA synthesis is necessary.
It appears that new mRNA synthesis is not essential for resumption of protein synthesis during the first hour of imbibition, which suggests that mRNA conserved in the dry embryo is utilized for early protein synthesis. It has also been proved that even if new mRNA is synthesized as an early event, it is not involved in early protein synthesis.
Ribosomal RNA (rRNA) synthesis commences as early as mRNA but increases during and after imbibition, particularly after the 6th hour coinciding with the time of radicle elongation and at 16-18h.it reaches a rate about 12-fold greater than that at the earlier stage.
Although transfer RNA (tRNA) synthesis begins within 20 minutes in imbibing embryos it is not certain whether this is involved in early protein synthesis, it can be presumed that tRNA is present in the dry embryo in excess of its requirement for protein synthesis during imbibition, whereas its new synthesis is necessary during later stages to sustain-growth.
The radicle within the seed initially grows by cell elongation and emergence through the seed coat may be associated with cell division. DNA synthesis which is closely linked to mitotic cell division is mainly concerned with the post-germination period of seedling development.
In wheat embryos, DNA replication occurs after radicle expansion, about 15 h after the initial hydration of the embryo.
DNA polymerase appears to be synthesized de novo during germination for if protein synthesis is inhibited during the first 9 h, the subsequent DNA synthesis is inhibited Inhibition of protein synthesis after 9 h is less effective in preventing DNA synthesis, indicating that the required polymerase is synthesized before the 9th hour.
Biochemical Change # 3. Mobilization of Stored Reserves:
There are two catabolic pathways of starch. One is hydrolytic and two amylases are involved:
In dicot seeds, starch degradation yields more glucose and maltotriose than in cereals, where more maltose is produced. This is related partly to the relative activity of cc-amylase in the two classes of seeds.
The other catabolic pathway is phosphorolytic:
Sucrose is the major form in which the products of carbohydrate catabolism are transported into the developing seedling.
The following reactions take place toward sucrose synthesis:
The phosphate group is split off from sucrose-6-phosphate by sucrose phosphatase. When necessary, sucrose can be hydrolyzed to free glucose and fructose by a α-fructofuranosidase (e.g., sucrase and invertase). Three discrete bodies found within fat-storing cells of seeds are involved in the breakdown of fat reserves. These are fat-storing oil bodies, the mitochondrion and the glyoxysome.
These three organelles function in the following manner:
(i) Lipolysis by lipase to give fatty acids and glycerol occurs in the oil bodies
(ii) oxidation of fatty acids and synthesis of succinate via the glyoxylate cycle takes place in the glyoxysome
(iii) Conversion of the succinate to oxaloacetate is done by the mitochondrion. The oxaloacetate undergoes further reactions in the cytoplasm and ultimately yields sucrose
Storage proteins of seeds are hydrolyzed into their constituent amino acids by proteinases or proteases.
These enzymes can be classified based on the nature of their substrates:
(i) Endo Peptidases:
These enzymes cleave the internal bonds of the polypeptides to form smaller polypeptides.
(ii) Amino Peptidase:
These enzymes sequentially cleave the terminal amino acid from the free amino acid end of the polypeptide chain. These are exopeptidases.
(iii) Carboxy Peptidases:
These enzymes sequentially cleave single amino acids from the carboxyl end of the polypeptide chain. These are also exopeptidases. Another class of hydrolyzing enzymes are there which hydrolyze various small peptides but not proteins; these are peptide hydrolases.
Thus, the hydrolysis of proteins to amino acids may take place along two pathways:
