Based on the results of this experiment, using of chitosan nanoparticles spraying, during rice grain growth and development (spike onset, flowering onset and milky onset of grain), resulted in reduced total protein, total weight of amino acids, especially phenyl level in the rice kernel compared to control treatment.
Of the most important reasons for the metabolic response of rice grains to the effect of chitosan nanoparticle spraying, is the role and importance of the molecular structure of chitosan nanoparticles. Chitosan nanoparticles were able to interfere with amino acid synthesis pathways due to their active functional groups (OH, H, NH2, O) and altered the enzymatic processes of amino acid synthesis. Past researches has shown that chitosan nanoparticles have the ability to inactivate and stabilize the enzymes that interact with them . The results of previous research also showed that chitosan nanoparticles are effective in changing pathways and the result of amino acid synthesis process  .
In this experiment, the effect of chitosan nanoparticles on the types of amino acids produced in the rice kernel was not uniform. For instant, under the influence of one concentration like 1000 µl L− 1 of the chitosan nanoparticles spraying, some amino acids decreased (e.g., the Phe) and some increased (e.g., tyrosine), and others remain unchanged. The increasing or decreasing effect of chitosan nanoparticles on the amount of amino acids in the kernel depends on the reactivity of the amino acids precursors, the enzymes of the synthesis pathway, and the substrate level of the enzymes.
For example, some steps of the metabolic reaction are similar in the synthesis pathway of some amino acids such as phenylalanine and tyrosine, and only in the late steps of the synthesis pathway, the reactions are difference . Because they should bind to a particular enzyme or a particular substrate, until to produce a different product. Thus, any factor that interfere with the synthesis steps and bind to the substrates or enzymes associated with the terminal steps of the synthesis pathway may play a role in determining the type of amino acid and its rate of production, and even in the aberration and stopping of amino acid synthesis pathway . Therefore, based on the results, weight loss of total amino acids produced in the rice kernel, especially the significant reduction of the Phe, could be due to changes in the enzymatic reactions which are related to the amino acid synthesis pathways.
The results showed that the amount of production of one type of amino acid varies at different concentrations of chitosan nanoparticles. Indeed, the effect of chitosan nanoparticles is related to changes in the nanoparticles concentration. It means that, the effect of varying the concentration of chitosan nanoparticles on the synthesis and production of an amino acid has different effects.
One of the most important causes of different effects (changes in the concentration of chitosan nanoparticles) on the production of an amino acid, is due to the effect of changing the total number of functional groups present in the molecular structure of the chitosan nanoparticles  . Changing the total number of functional groups of the molecular structure of chitosan nanoparticles affects the reaction efficiency of chitosan nanoparticles, because they are functional groups of chitosan nanoparticles that interact with the amino acid synthesis pathway as effective and reactive agents . Therefore, the participation rate of the functional groups is as an effective factor in guiding the reactions of the amino acid synthesis pathway and determining the product type of the synthesis process . In other words, the number and types of reactive functional groups influence the choice and preference of the reaction of chitosan nanoparticles with a variety of regulator factors in the amino acid synthesis pathway.
In addition, other factors such as plant species, environmental conditions (temperature, soil moisture and pH), plant physiological growth stage, method of nanoparticle application, timing, and amount of chitosan nanoparticles can have different effects on the performance of chitosan nanoparticles  . Therefore, the effects on plant growth and the metabolic response of the plant will not be uniform. Because amino acid synthesis reactions in plants are sensitive to regulators of amino acid biosynthetic pathways .
The other most important results in this study were the increased reaction of the PAL activity under the influence of the spraying application of chitosan nanoparticles in the rice kernel during grain growth and development stages. Although in all concentrations of chitosan nanoparticles, activity of the PAL was higher than the control treatment, the maximum activity of the PAL obtained with 1000 µl L-1 of chitosan nanoparticles.
The increased activity of the PAL under chitosan has also been reported in other previous studies  and is in agreement with these results. Past researches also reported that by using spraying chitosan nanoparticles as an exogenous elicitor on plants, they enhanced the plant's defense system through stimulating the activity of defense and antioxidant enzymes and enhanced plant resistance to attack by pathogens . In fact, plants affected by chitosan nanoparticles enhance the defense mechanism of the synthesis of antioxidant enzymes such as superoxide dismutase, catalases and peroxidases, as well as the production of phenylalanine ammoniumase and other plant phytoaloxins and secondary metabolites .
The results of a study on the reduction of the PAL activity suggest that compounds such as p-Coumarate, o-chlorosinamate, p-benzoate, 2-nafhtholite and trans-cinnamate that are in place of the PAL can bind to the enzyme, then results in disruption and/or reduction of the phenylalanine ammoniasis activity . For example, increasing the trans-cinamat level in the place of the PAL enzymatic can compete with the Phe for binding to the PAL, as a result inhibit the enzymatic activity of the PAL on the Phe. In fact, this inhibition does blockage metabolic pathway of the Phe for conversion to the other phenolic compounds . However, this type of inhibition of the PAL effect on the Phe is not always inappropriate especially when plants are exposed to inappropriate changes in environmental conditions and biological and non-biological stresses to maintain plant life, the normal metabolic reactions and biosynthetic pathways of amino acids change temporarily, and these changes are the same plant responses to the effects of biological and abiotic stresses.
On the other hand, the activity of the PAL also depends on the amino acid concentration of phenylalanine, meaning that at lower concentrations than the Phe, it will have more enzymatic activity and this type of reaction is a result of the specificity of the the Phe accumulation, and as the feedback inhibitory cycle of the Phe is well known and it can affect the PAL activity .
Also, the results indicate that based on the quantities of essential amino acids produced in the rice kernel, the trend of changes in essential amino acids production was not uniformly under the spraying of chitosan nanoparticles and they had different reactions to each other. For these differences, it could be argued that the potential differences in the structure of molecules and compounds present at the site of metabolic reactions along with the functional groups of chitosan nanoparticles could be due to differences in amino acid production.
For example, according to the results of Table 4, amino acids that were raised to a concentration of 1000 µl L− 1 concentration of chitosan nanoparticles were structurally first of non-polar structures (e.g., alanine, isoleucine and valine), and later of polar structures and neutral (e.g., tyrosine and threonine). But, the amino acids that were reduced at 1000 µl L− 1 concentration of chitosan nanoparticles were more of the non-polar structures (e.g., phenylalanine, methionine, and tryptophan), followed by the negatively charged polar types (e.g., glycine and aspartic acid). Interestingly, the amino acid phenylalanine is a nonpolar and cyclic structure that is reduced by chitosan nanoparticles, and in contrast, tyrosine, it increases by a neutral and cyclic polar structure by chitosan nanoparticles. Therefore, the results show that in the first step, the polarity of molecule and, secondly, the electrical charge of the molecule determines the potent of reaction of compounds with chitosan nanoparticles at the site of metabolic reactions.
This kind of reaction between chitosan nanoparticles and other compounds, e.g., amino acid precursors, seems reasonable because, first, the molecular structure of chitosan nanoparticles affected by the synthesis method, and second, the type and number of functional groups in the structure of chitosan nanoparticles, and third, the lack of an equal reaction of each of the functional groups due to the differences in the type and molecular structure of the substrate compounds in the amino acid biosynthesis pathway, which allows the reaction to be preferred and to decrease or increase the amino acid production. Therefore, the effect of chitosan nanoparticles spraying on the rice grain will result in a difference in amino acid production in the rice kernel.