INDUCTION OF NITRATE REDUCTASE BY LIGHT AND NITRATE
INTRODUCTION
The most common source of nitrogen for most plants is nitrate. Nitrate is absorbed and reduced first to nitrite and then to ammonium, either in roots, shoots, or both, depending on species and environment. Ammonium ions are subsequently converted to glutamic acid and then to all other essential organic nitrogen compounds.
The first step in converting nitrate to ammonium is catalyzed by nitrate reductase (NR). NR uses NADH to reduce nitrate (NO3- ) to nitrite (N02- )
NO3- + NADH ----> N02- + NAD + H20
NR is an interesting enzyme because of its critical importance in nitrogen metabolism and because it can be induced by certain environmental conditions. Plants grown without nitrate (i.e., with ammonium ions instead), do not need NR so it is usually found in almost undetectable amounts in both roots and shoots. Addition of nitrate to such plants is normally followed by large increases in NR activity due to synthesis of the enzyme rather than conversion of an inactive protein to an active form.
Although the presence of nitrate seems sufficient for normal NR synthesis in roots, light is also needed for NR synthesis in leaves. For example, very little NR is detected in the leaves of corn seedlings grown several days in darkness, even in plants watered with a solution containing nitrate. Exposing such plants to several minutes of light induces the enzyme after a lag period of several hours. These dark-grown plants had a limited capacity to synthesize proteins. Light promotes translation, leading to increased production of many proteins including NR. It is likely that red light, acting through phytochrome, is responsible for this stimulation.
In addition to light-induced translation,, nitrate reduction is further stimulated by another light effect. It has long been known that green leaves exposed to adequate light contain less nitrate than leaves shaded for several hours, even though both contain enough nitrate for NR induction. This results from the production of NADH (needed for NR activity) during photosynthesis, and may not involve NR induction. The shaded leaves simply do not produce enough sugars for the rapid production of NADH in subsequent metabolism. Adequate light increases photosynthesis and export of Calvin cycle intermediates to the cytoplasm. Leaf NR is localized in the cytoplasm, and glycolysis may be the main source of NADH. The subsequent conversion of nitrite to ammonium occurs in the chloroplast in leaves; the location in roots is not established.
In this experiment you will study the effects of light and nitrate on activity of NR in leaves of corn, mung bean, barley and soybean seedlings.
PROCEDURE
A. NR Preparation
1. Collect 1gram of leaves from plants grown for one week as follows
a. 7 days in darkness in plus-nitrate solution.
b. 6 days in darkness in plus-nitrate solution, then 1 day in continuous light
c. 7 days in darkness in minus-nitrate solution.
d. 6 days in darkness in minus-nitrate solution, then 1 day in continuous light
2.. Chill the leaves in ice water and perform all subsequent steps using ice cold solutions and glassware.
3. Slice the leaves finely with a razor blade on a glass plate. Grind them in 6 mls grinding buffer. Use a cold mortar and pestle, and surround the mortar with ice.
4. When the tissue is completely homogenized, transfer the brei to a centrifuge tube. Rinse the mortar & pestle with 4 mls buffer and add to the brei, then spin at 27,000x g for 15 min at 4˚ C.
5. Transfer the supernatants to fresh plastic tubes and keep on ice
B. NR Assay
You will be given a premixed buffered substrate solution (15 mM phosphate and 10 mM potassium nitrate) and an NADH solution (2.5 mM). Use 15 ml plastic centrifuge tubes for the assays.
1. Set up 2 tubes containing 4 ml buffered substrate solution and 0.5 mls NADH. Start the reaction by adding 1 ml extract (save the left-overs and keep on ice!). Mix well and incubate at 30˚C for 30 min. Also prepare a control tube with 1ml grinding buffer instead of extract.
2. After 30 min. stop the reactions by adding 250 µl of l M zinc acetate; mix, then centrifuge at 10000 x g for 2 min.
3. Carefully remove the supernatant with a pasteur pipette. Place the supernatants in fresh tubes. The supernatant must be clear; if not, recentrifuge.
4. Add 250 µl of 0.3 mM phenazine methosulfate (to destroy excess NADH) to the supernatant, mix, and incubate at room temperature for 20 min in darkness since phenazine methosulfate is light-sensitive.
Color Development
Now you will use a “color reagent” (a commercial reagent for detecting nitrite in aquarium water) to determine how much nitrite is present in each tube.
1. Transfer 4 mls of each reaction to a Spec 20 tube. Save the left-overs and keep in the dark.
2. Add 200 µl color reagent to each Spec 20 tube, mix, and store at 22˚C for 5 min. The color reagent is light sensitive, so keep the reaction tubes in the dark.
Standard Curve
l . Prepare 4 mls of 0, 10, 20, 30, 50, 70 and 100 nM nitrite solutions in Spec 20 tubes using RO water and a stock solution of 1 µM sodium nitrite.
2. Add 200 µl color reagent to each Spec 20 tube, mix, and store at 22˚C for 5 min. The color reagent is light sensitive, so keep the reaction tubes in the dark.
3. Use the 0 nM tube to blank the spec 20 at 540 nm. Read the A 540 for the standards, and then for each sample.
4. If necessary, prepare suitable dilutions of the samples to get the reactions “on scale.” Mix suitable amounts of the reactions from step 4 of “NR Assay” with water to give a total volume of 4 mls, then add 200 µl of color reagent. Be sure to keep track of the dilution factors!
5. Use the standard curve to determine the nanomoles of nitrite produced in each experimental tube. Don’t forget to consider the result of the no-enzyme blank! You have now determined the net rate of reduction by 1 ml extract/ 30 minutes.
6. Convert the rate of reaction to nM nitrite/gram tissue/hour.
Write up
Your introduction should explain why nitrate reductase is important, why we might expect it to be induced by light and nitrate, and that the purpose of this experiment was to see whether nitrate reductase was induced by light and/or nitrate in any of the tissues studied.
Your materials and methods should succinctly describe the procedure followed. Your results should state the purpose of the experiment, state that the raw data are presented in Table I and the processed class data are presented in Table II, and should point out the key features. Did any plants have nitrate reductase activity? Were there differences between plants?
Your discussion should explain the results. Why were there differences between plants (if any)?