Nitrogen is an essential element of all amino acid synthesis and components of nucleic acid and chlorophyll. The plant uptake the nitrogen from soil mainly with the nitrate form. However, the exogenous nitrate can be assimilated by an enzyme into the organic product. According to Srivastava (1979), the nitrate reductase (NR) is one of the most important enzymes to reduce nitrate to nitrite, then the nitrate is going to a complex pathway to NH4+ and to amino acid. So there is positive correlation between NR activity and grain protein and nitrogen. The NR reduce nitrate to nitrite using the NADH as the electron donor in cytosol, the nitrite transfer to chloroplasts of leaves is reduced to ammonium ion (NH4+) using ferredoxin as the electron donor.
Materials and Methods
The specimen in this lab is the Hordeum sativum which the seeds are fed nitrate, ½ the concentration nitrate once a week, and provided light, compared with the fed normal nitrate with 48 hr dark and the unfed seeds in the light. Before 5 hr the assaying, the 10 samples tube and one blank with 4 leaves and 4 roots are prepared with 0.5g and incubated with the 12ml incubation solution (0.25M KH2PO4,0.05m KNO3,20% v/v,2-propanol), and 6ml incubation solution into blank into the dark chamber and 1 fed leaves and 1 fed root into the light chamber. After 5 hr incubation, transferring 2ml from each tube into the test tube and adding 2.0ml sulfanilamide (0.1%w/v) and 2.0 ml NED(0.02%w/v) to each test tube, the 1-8 test tube are read and record the absorbance by spectrophotometer. Building the standard curve for nitrite, the six tubes with different amounts of nitrite are added 2.0 ml of sulfanilamide and 2.0 ml of NED and recorded the absorbance after 10 mins.
NR activity of FED sample = nmoles NO2– per gram of tissue per hour
= 20.47nmol/ 0.083g (2hr)
Table 1. The Hordeum sativum leaves and root nitrate reductase activity in 10 conditions
|Tube #||NO2– produce (nmol NO2–/G/hr)|
Basing on the equation from the standard curve, the absorbance and the amount of nitrite is directly proportional with the slope of 0.0082 and intercept of -0.0004. From the equation, the nitrate produce per unit with nmol/g/hr of 10 samples tested, the leaf always produce higher amount nitrite than root in the same condition, other than the two tubes in the light chamber. Furthermore, the leaf and root in the FED condition of dark chamber can produce the most amount of nitrite.
In this experiment, the purpose is that the barley plant ( Hordeum sativum) is effected by the activity of nitrate reductase within different nitrate feeding regimes. In general, the nitrogen support the plant growth, protein synthesis and production. The plant can uptake the nitrogen in the form of nitrate ion contained in the soil organic matter (Robert, 2016). The nitrate process a serious of reaction assimilate into organic compound which is necessary of plant. The pathway of nitrate assimilation are convert the nitrate ion to nitrite ion in cytosol, and the nitrite ion is transferred to chloroplast in green tissue of plant, or the plastids in non-green tissue. Then, the nitrite is reduced to ammonium ion and the ammonium change to amino acid in the same place of different tissue. Additionally, the nitrate reductase, nitrite reductase and glutamine synthetase as the significant enzyme to participate in the reactions. However, the barley plant in the lab grow in a vermiculite/perlite meaning that no nutritional content and control to feed the nitrate and artificial light in vivo at 4 different condition within 3 weeks. The test condition is that the incubated plant in tube by taking in the nitrate in the solution.
In terms of the Buczek (2015), the light stimulate the nitrate assimilate within the cotyledon, and depresses it in the roots. Responding to the result, the test tube #1, 2 with light is higher production of nitrite than the #5, 6 with 48hr dark. The light effect of plant can be recognized to photosynthesis. Referring to reactions of nitrate assimilate, the NADH as the electron donor is oxidized to NAD+, and the nitrate ion accept the electron to nitrite ion by using nitrate reductase, and the ferredoxin donor the electron to reduce the nitrite ion to ammonium ion in chloroplast or plastic as well as the ammonium ion into the amino acid by glutamine synthetase in chloroplast of green tissue.
The incubation solution has two main reagents: monopotassium phosphate, potassium nitrate. Monopotassium phosphate helps reduce the loss of the ammonium ion, by maintaining the pH at a slightly low level. Potassium nitrate improves the capacity of roots to take up nutrients and solutions. Further, it improves the integrity of the tissue structure of the plant once it is assimilated with glucose in the leaves to form proteins.
In most environments, plants get a specific amount of hours of sunlight, based on the season (Nunes et al., 2011). It was with this knowledge in mind that even the light set up was made in such a way that it did not allow for round-the-clock exposure. This set up made it easy to evaluate the results in vivo as accurately as possible.
In the light reaction, the water serves as the electron donor and NADP, which receives an electron from the breakdown of chlorophyll to become NADPH, is the acceptor. The water is broken down yielding electrons that are then received by the NADP during the photosynthetic processes. For dark reaction, NADPH serves as the donor, releasing an electron, while, NADP remains the acceptor.
The coupling of photosynthetic electron flow and the reduction of nitrite is evident at the ferredoxin level, explaining why there is no assimilation of nitrates in the dark (Zhao et al., 2015). For the plants in the light room, the electron flow follows the natural pattern leading to the addition of an electron to ferredoxin, allowing the process to continue to the next stage in the nitrate assimilation process (Zhao et al., 2015).
In this experiment, the light reaction takes place in the thylakoid membranes, where pigments such as green chlorophylls are combined with proteins and electron carriers, to make photosystems, which are used to harvest light. The dark reaction takes place in the Stroma, where the energy generated by the ATP and electrons from NADPH add the CO 2 into carbohydrate.
The CO2 is then compounded with ribulose bisphosphate to generate a six-carbon molecule, which then splits to two set of three-carbon molecules. These two molecules eventually regenerate ribulose bisphosphate and glyceraldehyde phosphate. Glyceraldehyde phosphate then moves on to form glucose or other sugars, which are mainly stored in plant stems and grains, for this species (Zhao et al., 2015).
For the experiment the enzymes reside within the plant tissue and by pairing with the reagents in the incubation solution and based on the availability of other factors such as light, they help in the breakdown of various components.
The membrane-bound nitrate reductases (Nar) are structures comprising three different subunits. The NarG and NarH subunits are found in the cytoplasm, and positioned on the membrane by the NarI subunit. The narG-encoded catalytic subunit carries the active site, a guanylyl molybdenum cofactor, and one [4Fe-4S] iron-sulfur cluster. The narH-encoded subunit is contains one one [3Fe-4S] iron-sulfur cluster and three [4Fe-4S] iron-sulfur clusters (Tian et al., 2011).
The regulation of nitrate reductase is mainly a function of stress, salinity and for this experiment, the role was well served by the introduction and elimination of. Nitrate reductase is the main control point of the nitrate assimilation channel and it is regulated at transcriptional, translational and post-transcriptional levels. The availability of light and oxygen are the most important. Transcription is boosted by the presence of nitrates and light. Nitrite Reductase has also been found to be regulated in the post-translation level through phosphorylation, with light having been found to greatly influence mRNA translation and the stability of proteins.
The fastest way a plant can regulate Nitrate Reductase in vivo is by controlling the speed at which NADP is converted to NADPH. An increase in NADPH increases the NR activity as has been shown by this experiment, where the samples conducted in light confirmed that the absorbance and the amount of nitrite produced is directly proportional.
From this experiment, the end point of the reactions is different from how it happens in natural conditions on the basis of the fact that in the lab, the nutrients were added without consideration of the quantity of other elements such as enzymes in the samples. In natural conditions, the nutrients and the entire breakdown process are regulated to factor in the availability of different food production elements. In this experiment, the end point was arrived at much earlier than in natural conditions because of the addition of incubation solutions.
Carbon and nitrogen metabolism are interlinked because both elements serve as dampening grounds for ATP, hence reducing the energy produced during photosynthesis (Tian et al., 2011). Similarly, the intermediates of both Carbon and Nitrogen metabolism are necessary for most critical plant functions such as photosynthesis.
Nitrogen accumulation in barley mainly happens in the seeds/grains but during the developmental stage, it is stored in all tissues from the stems to the leaves. Through the application of fertilizers and other components containing nitrogen and elements like phosphates, the amount of nitrogen in grains can be regulated. Farmers, whose produce fetches the best prices usually ensure that they have optimum amount of nitrogen-provoking additives provided to the plants during the production phase.
This experiment was conducted to show the importance of Nitrogen in plants and it has shown so through noting the absorbance of Nitrites, ultimately proving that there is a correlation between NR activity and grain protein accumulation.
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Zhao, J., Qiu, Z., Ruan, B., Kang, S., He, L., & Zhang. S. (2015). Functional Inactivation of Putative Photosynthetic Electron Acceptor Ferredoxin C2 (FdC2) Induces Delayed Heading Date and Decreased Photosynthetic Rate in Rice. PLoS ONE, 10 (11), 1-77.