NITRATE REDUCTASE ACTIVITIES (NRA)

By :
Sesti Wan Tiyah B1B018025
Intan Khoerunnisa B1B018037
M. Fuad F B1B018043
Ana Fatimatus S B1B018052
Eka Restiyana B1B018055
Rombongan : VIII
Kelompok : 5
Asisten : Devi Vira Setiana

PLANT PHYSIOLOGY I LABORATORY REPORT

MINISTRY OF RESEARCH, TECHNOLOGY, AND HIGHER EDUCATION

JENDERAL SOEDIRMAN UNIVERSITY
FACULTY OF BIOLOGY
PURWOKERTO
2019
 I. INTRODUCTION

A. Background

Nitrate reductase is an enzyme that catalyzes the reduction of NAD(P)H from

nitrate to nitrite. Nitrate reduction that occurs before amino acids are produced
requires electrons. NR is found in plants, algae, and fungi that function for nitrogen
metabolism. NR is a type of flavoprotein that has a co-enzyme in the form of FAD
(Flavin Adenin Dinucleotide) and has cytochrome b-557 and has MoCO
(Molybdenum Cofactor). The mechanism of action of NR is an inducible enzyme
induced by the substrate. Other factors such as CO 2 levels, light intensity, pH,
fertilizer, salinity and glucose levels (Baraba´s et al., 2000).
NR levels also correlate with nitrate levels in plant organs. This is because
these enzymes are widely distributed in various plant organs such as leaves, flowers,
roots, stems, and fruit. The spread of NR between organs varies from one plant to
another. NR levels are mostly found in leaves, then stems, roots, fruits, and the
smallest are found in seeds. However, the spread of NR in plant organs can also be
influenced by many factors such as the availability of the substrate, plant type, plant
age, and nutrient conditions (Marschner, 1995).
Peanut (Arachis hypogea) originates from Latin America and develops to
Asian countries such as India, the Philippines, Japan and Indonesia. In Indonesia,
according to the results of the Research Institute for Nuts in Bogor, there are already
4 types of superior varieties, namely varieties of Elephant, Bull, Tiger and Deer.
Deer varieties have the largest oil content, which is 49.9 percent of the weight of
meat (Ketaren, 1986). Classification of peanuts (Arachis hypogaea), Kingdom is
Plantae or herbs, Division is Spermatophyta or seed plants, Subdivisions is
Angiosperms or covered seeds, Class is Dicotyledoneae or seeds in pieces, Order is
Leguminales, Family Papilionaceae, Genus Arachis and Species is Arachis
hypogeae L (Peni, 2015).
Peanut (Arachis hypogaea L.) is economically a legume crop that ranks second
after soybeans, so it has the potential to be developed because it has high economic
value and considerable domestic market opportunities. Peanut seeds can be used
directly for food in the form of vegetables, fried or boiled, and as industrial raw
materials such as cheese, soap and oil, as well as stover for animal feed and fertilizer
(Marzuki, 2007).
B. Objectives

The objective of this laboratory activity is to find out the activity of nitrate
reductase in peanut (Arachis hypogea) plants.
 II. REVIEW OF LITERATURE

Nitrogen is an important element in plant life. In addition, equally important

elements are carbon, hydrogen, and oxygen as constituents of proteins, nucleic acids,
growth hormones, vitamins and others. Nitrogen in plants is in the form of amino
acids, proteins, amides, chlorophyll, alkaloids, and nitrogen bases. The leaves consist
of 1-25% nitrogen by its dry weight, but the percentage of nitrogen is less present in
other vegetative organs. Nitrogen cannot be used in the form of NO 3- or NH4+ but
must be converted into amino gases and proteins (Harahap, 2012).
Metabolism is one of the most important aspects for plant and animal life.
Nitrogen metabolism can be defined as a series of biochemical processes which take
place inside or outside the body of the plant in the form of the formation of nitrogen
complexes from simple molecules and the overhaul of the nitrogen complex into the
simple molecules that make it up. Based on this understanding, nitrogen metabolism
includes anabolism, which is formation, and catabolism, which is the process of
reshuffle. Important anabolic processes include nitrogen fixation, amino acid
synthesis, and protein synthesis, and catabolism processes including proteolysis,
amino acid reshuffle, denitrification, and nitrification. The process of photosynthesis
is also an anabolic process whereby when a bright reaction produces ATP and when
a dark reaction occurs the binding of carbon monoxide gas is accompanied by the
formation of carbohydrates (Pandey & Sinha 1990).
The activity of a number of enzymes from various plant metabolic pathways
is regulated by light. Nitrate reductase is a regulator of nitrogen assimilation in
plants, which is regulated by changes in light / dark. Although the response of nitrate
reductase activity to light / dark can vary from species to species, in general its
activity is higher in light conditions than dark. Light increases nitrate reductase
activity by accelerating nitrate uptake (Latifa, 2009). Nitrate reductase is mediated by
nitrate reductase which produces nitrite. Nitrite will then be converted to ammonium
through nitrate reductase. In addition to assimilation, nitrate reductation acts as a
synthesis of nitric oxidation, which is a molecule that is recognized as a signal of
transduction in plants (Schoenbeck et al., 2015).
An enzyme is a protein compound that is a catalyst in a reaction that is
produced through the expression of certain genes. Enzymes work specifically in
metabolic reactions. Almost all metabolic reactions are regulated and controlled by
enzymes. Likewise, metabolic regulation runs dynamically which involves the
interaction of DNA and RNA as well as the characteristics of each organism. Nitrate
Reductase (NR; EC 1.6.6.1-3) is an enzyme that catalyzes the reduction of NAD (P)
H from nitrate to nitrite. Nitrate Reductase is found in plants, algae, and fungi that
function for nitrogen metabolism (Campbell et al., 2008). NR is a type of
flavoprotein that has a co-enzyme in the form of FAD (Flavin Adenin Dinucleotide)
and has cytochrome b-557 and has MoCO (Molybdenum Cofactor) in its mechanism
of action. Nitrate Reductase is an inducible enzyme induced by its substrate, nitrate
(Munzarova et al., 2006).
Nitrate reductase is the main enzyme found in photosynthetic active tissue.
Nitrate reductase has a very important role in the initial process of amino acid
synthesis. Nitrate reductase biosynthesis depends on the availability of nitrogen
nutrients and its activity can be induced by the presence of nitrates in the leaves
(Primavani & Zulaika, 2014). Nitrate Reductase is an enzyme that was first used in
the nitrate assimilation pathway, which converts nitrates to nitrites with NADH /
NADPH as the electron donor. In general, nitrate assimilation occurs in the leaves
although some plants such as soybean do nitrate assimilation in the roots. When
nitrate (NO3-) is absorbed by the roots, then nitrate reductase will reduce nitrates to
nitrites and then nitrites are transferred to leukoplas to be reduced to ammonia.
Nitrates will also be transported to the leaves via xylem vessel bundles. Here nitrate
is also reduced to nitrite by nitrate reductase and the product will be reduced to
ammonia in chloroplasts. Nitrates can also be stored in vacuoles. Because nitrate
reduction occurs in the cytosol, at any time nitrates in vacuoles can be transferred to
the cytosol to be reduced (Heldt, 2005).
The level of molecular regulation of the nitrate reductase enzyme is
controlled by the nitrate reductase-producing gene. The nitrate reductase gene can be
induced by nitrate, light, and glucose. While nitrate reductase gene inhibitors are in
the form of ammonia and glutamine. After the nitrate reductase gene synthesizes the
nitrate reductase enzyme, the nitrate reductase enzyme will be activated by a series
of protein kinases through the nitrate reductase kinase pathway. The protein kinase
will be phosphorylated so that the nitrate reductase enzyme becomes active. In
addition, Ca2 + ions can also stimulate the activation process of the nitrate reductase
enzyme. Nitrate reductase enzymes also have inhibitors such as protein inhibitors,
oxalic acid, triose phosphate, and other types of phosphate esters that are able to
deactivate the nitrate reductase enzyme (Heldt, 2005). In addition to the nitrate
reductase gene involved in the synthesis of the nitrate reductase enzyme in nitrate
reduction, there is another gene that acts as a substrate transporter of the nitrate
reductase enzyme, the NRT2 gene (NR Transporter). The NTR2 gene will synthesize
the protein transporter so that the substrate in the form of nitrate can enter the cell.
This NTR2 gene can be inhibited by glutamine which is the result of changes from
ammonia. Ammonia can also inhibit nitrate transportation into the cytosol (Jackson
et al., 2008). In the biogeochemical cycle, the enzyme nitrate reductase plays an
important role in the process of nitrification or nitrite formation from nitrates. The
next stage is the product of nitrate reductase, nitrite will be ammonified with the help
of the enzyme nitrite reductase (NiR). With the help of the nitrogenase enzyme,
ammonia will be converted into free nitrogen gas (Voet, 2009).
Nitrate reductase is an important enzyme in the reduction chain of the
element nitrate to ammonia which is useful in the formation of amino acids, protein,
chlorophyll and other compounds containing nitrogen. Nitrogen in the atmosphere is
in the form of N2. A small portion of N2 will oxidize by O 2 to nitrogen oxide (NO,
N2O). Nitrous oxide will then undergo oxidation of acids to nitrates which, trough
rainwater, will enter the ground. Other sources of nitrogen in the atmosphere are
ammonia (NH3-) and ammonium ions (NH4+) from industrial combustion, volcanic
activity and forest fires (Anggarwulan & Solichatun, 2001). Absorption of nitrate
and ammonium ions allows plants to form various protein compounds (Salisbury &
Ross, 1995).
The consersion of organic nitrogen into ammonium ions by bacteria and soil
fungi is called ammonification. The ammonium ion is then oxidized to nitrite and
nitrate. This oxidation is called nitrification. Nitrates undergo further changes into
NO and N2O through stages called denitification (Guleryuz & Arslan, 1998).
Most nitrates are absorbed by the roots, while nitrate assimilation from most
tall plants occurs in the leaves. Nitrates, before being assililated in the leaves or
roots, must be reduced to ammonia. Nitrate reduction in tall plants is divided into 2
reactions. First the nitrate is reduced to nitrite which is catalyzed by nitrate reductase
(NR), the second reaction is the conversion of nitrate to an ammonium ion catalyzed
by nitrate reductase (NR). In the first reaction, the atom is released as water. The
reaction from nitrate to nitrite can be written as follows (Noogle & Fritz, 1983).
 Nitrate reductase activity is influenced by several factors, including the rate
of synthesis and the rate of overhaul by by protein-destroying enzymes and inhibitors
and activators in cells. Light can increase nitrate reductase activity if nitrate is
available which causes daily rhythm (day and night) in the activity of nitrate
reductase. During water stress, nitrate reductase activity drops rapidly. This is due to
a decrease in the movement of nitrates to the leaves as a result of reduced
transpiration (Salisbury & Ross, 1995). Other factors that influence nitrate reductase
are the influence of PH, temperature, plant age, and inhibitors. The effect of PH is
that if an enzyme shows activity at a certain PH goes down or rises it that if an
enzyme, therefore in measuring nitrate reductase activity a Na-phospate buffer
solution is used to balance the PH. Each enzyme also has a different optimum PH.
Temperature is low, the enzyme is inactive and when the temperature is high, the
enzyme is denatured (Gardner, 1991).
The plants are young, nitrate reductase activity will increase and reach its
maximum, then it will decrease during aging. The presence of inhibitors that
interfere with photosynthesis and respiration reactions, such as high light intensity
will reduce the activity of nitrate reductase, because the reducing energy needed by
enzymes is distrurbed by its formation, In the biogeochemical cycle, the enzyme
nitrate reductase plays an important role in the process of nitrification or nitrite
formation from nitrates. The next stage is the product of nitrate reductase, nitrite will
be ammonified with the help of the enzyme nitrite reductase. With the help of the
nitrogenase enzyme, ammonia will be converted into free nitrogen gas
(Bidwell,1979).
1.
 IV. RESULT AND DISCUSSION

A. Result

Tabel 4.1 Nitrate Reductase Activities of Leaf

No Type of Fertilizer Absorbance Nitrate Concentration
1 Urea 0.203 1.80
2 Compost 0.140 0.83
3 KNO3- 0.168 1.28

Grafic 4.1 Nitrate Reductase Activities of Leaf

Calculation:

Urea 0.203 KNO3- 0.168

Kompos 0.14
Figure 4.1 Nitrate
Y = 0.0651x + 0.0854 Reductase Activities of Y = 0.0651xY+=0.0854
0.0651x + 0.0854
Leaf
0.203 = 0.0651x + 0.0854 0.14 = 0.0651x = 0.0651x +
+ 0.0854
0.168
0.0854
X = 0.1176 X = 0.0546
0.0651 0.0651 X = 0.0826
0.0651
= 1.80 =0.83
=1.26
 III. MATERIALS AND METHODS

A. Materials

The tools that used in this laboratory activity are erlenmeyer/ bottle that is
given a covery/ styrofoam, analytic scale, scissors and gardening scissors, ruler
and label.
The materials that used in this laboratory acivity are the cutting of Arachis
hypogaea plants, water, 0,1 M phosphate buffer, sulfanil amide, 5 M NaNO3
solution, N- naphthlethylene diamine (NED) 0.02% and aquades.

B. Methods

The 3rd Arachis hypogaea leaf from the top

Taken, washed, sliced (eliminated the bones)

Weighed by 200mg

The phosphate buffer was put in 0.1 M pH 7.5 in volume 5 ml

incubated 24 hours

The buffer liquid was replaced with a new 5 ml

0.1 ml NaNO3 5 M is added

 Incubation for 3 hours

0.2 ml of Sulfanil amide and 0.2 ml of N-naphthyltilene diamine are added

0.1 ml of aliquots is added waited until the color change

2.5 ml of water is added until the total volume is 3 ml

Absorbance value is measured 540 nm waves of spectrophotometry

The result is written

B. Discussion

Nitrate reductase levels also correlate with nitrate levels in plant organs. This is
because this enzyme is widely distributed in various plant organs such as leaves,
flowers, roots, stems, and fruits, but the spread of nitrate reductase between one
organ to another is different in each plant. Nitrate reductase levels are most
commonly found in leaves and stems, roots, fruits, and the smallest are found in
seeds. The spread of nitrate reductase in plant organs can also be influenced by many
factors such as the availability of the substrate, plant type, plant age, and nutrient
conditions (Marschner, 1995). The work of the nitrate reductase enzyme can also be
influenced by other factors such as CO 2 levels , light intensity, pH, fertilizer,
temperature, concentration, plant age, inhibitor compounds, and salinity and glucose
levels (Baraba´s et al., 2000).
The activity of a number of enzymes from various plant metabolic pathways is
regulated by light. Nitrate reductase is an enzyme that regulates nitrogen assimilation
in plants, which is regulated by changes in light or dark (Latifa & Anggarwulan,
2013). Although the response of nitrate reductase activity to light or dark can vary
from species to species, in general its activity is higher in light conditions than dark.
Light increases nitrate reductase activity by accelerating nitrate uptake (Puranik &
Srivastava 1985). The activity of nitrate reductase is influenced by light by
increasing the rate of photosynthesis which will produce carbohydrates and NADH
needed for nitrate reduction, produced from these carbohydrates when they are
aspirated. According to Noggle & Fritz (1983), photosynthesis is closely related to
nitrate assimilation, namely the formation of nitrate ions (NO3-) to nitrite (NO2-) and
ammonia ions (NH3+) into amino acids. Nitrate reductase activity affects the
synthesis of amino acids.
The assimilation of N into organic molecules depends on the reduction of NO 3 -
by the enzyme nitrate reductase in plant tissue. Nitrate reduction, which must occur
before acidamino is produced, requires electrons. The main electron donor is
nicotinamide adenine dinucleotide (NADH), which is the result of photosynthesis.
Blazing light and high photosynthesis rate are conditions conducive to the activity of
the nitrate reductase enzyme. Nitrate reductase biosynthesis depends on the
availability of nitrogen nutrients in the media, and its activity is induced by nitrates
that are adherent to the leaves (Gardner et al., 1991).
For plants that cannot harbor N2, the main sources of nitrogen are NO3- and NH4
+
. Most of the plants absorb nitrogen in the form of NO 3- because NH 4 + is
immediately oxidized to NO 3 - by nitrifying bacteria. But the conifer community
and grass absorb some of the nitrogen in the form of NH4 + because nitrification is
inhibited by low soil pH or by tannins and phenol compounds. Nitrate assimilation is
a change in nitrate to ammonium or ammonia (Heldt, 2005). It has long been known
that the step limiting rate for nitrogen assimilation which is the reduction of NO 3- to
NO2- and catalyzed by nitrate reductase (NR), is highly regulated. Increasing the
amount and activity of NR causes a corresponding increase in nitrate reduction
potential and provides greater capacity for general amino acid synthesis, protein
synthesis or total nitrogen assimilation (Marquez-Queros et al., 2014). This
conversion stage involves several enzymes as catalysts. Stages of change can be said
as a nitrate reduction process, shown as follows (Heldt, 2005):
(Nitrat)
1. HNO3 + 2e- +2H+ HNO2 + H2O
Nitrit reduktase
(Nitrit)
2. HNO2 + 2e- +2H+ HNO + H2O
Hiponitrit reduktase
(Hiponitrit)
3. HNO + 2e- +2H+ NH2OH
Hidroksilamin reduktase
(Hidroksilamin)
4. NH2OH + 2e- +2H+ NH4 + H2O
Hidroksilamin reduktase
(Hidroksilamin)
5. NH2OH + 2e- +2H+ NH3 + H2O
In nitrate reduction there is a nitrate reduction enzyme. The nitrate reduction
enzyme is a flavoprotein that contains FAD (Flavine Adenine Dinucleotide) as a
prosthetic group. In plant roots the enzyme nitrate reductase reacts with NADH2 /
NADPH2, the FAD of the enzyme is converted to FADH 2. Then FADH2 is oxidized
by transferring electrons to the molybdenum ion which is also an important part of
the nitrate reductase enzyme, and from the molybdenum the electron is finally
transported to nitrate. This is the reason why Mo deficiency in soils causes nitrogen
accumulation in plants (Heldt, 2005).
According to Rubatzky & Yamaguchi (1998), high nitrogen levels can stimulate
vegetative growth of plants, but if excess can delay plant maturity. Nitrogen is an
essential element of plant constituents that determines the quality of plant organic
matter. Nitrogen is present in a variety of plant protein compounds, nucleic acids,
hormones, chlorophyll and a number of primary and secondary metabolite
compounds. Nitrogen is also essential for cell division, cell enlargement, and for
growth. Symptoms of nitrogen deficiency are seen slowly and are shown by the
presence of chlorosis in mature leaves. Nitrogen deficiency conditions also result in
the accumulation of anthocyanin pigments, decreased protein content, accelerated
periods of decreased protein content, accelerated flowering periods, and growth
inhibition. Nitrate is a compound consisting of one element of nitrogen atoms and
three elements of oxygen atoms. Nitrate is a form of nitrogen that plants use in their
metabolism. Plants need nitrates to develop leaves, stems, immune system and seeds.
Although the element gas nitrogen, or N2, makes up nearly 79 percent of the Earth's
atmosphere, it is chemically active and cannot be used by plants. Nitrogen must be
converted to a form of chemically active nitrate through a process called nitrogen
fixation before it can be needed by plants.
Urea is an inorganic fertilizer made from synthetic and non-natural ingredients,
containing 46% nitrogen. In contrast to urea fertilizer that only contains nitrogen
nutrients, compost which is an organic fertilizer contains more nutrients both macro
and micro needed by plants. Compost is mainly dominated by the presence of
elements nitrogen (N), phosphorus (P), calcium (Ca), potassium (K) and magnesium
(Mg). Compost is a fertilizer made from organic materials such as leaves, stems,
weathered twigs, cattle dung, and others. Compost as well as other organic fertilizers
not only provide nutrients for plants but can also maintain soil structure. Unlike
compost, urea fertilizer provides a kind of nutrients that plants need. Nitrogen in urea
fertilizer will be directly absorbed by plants. (Lingga, 2013). KNO 3 is a type of
chemical fertilizer with potassium and nitrogen content in it. KNO3 fertilizer is a
combination of elements N (nitrogen) and K (Potassium) in the form of K 2O.
Potassium contained in KNO3 has an effect as a counterweight to the situation when
the plant is excess nitrogen, element K can also increase the synthesis and
translocation of carbohydrates, thereby increasing cell wall thickness, stem strength
and increasing sugar content (Foth, 1994).
A spectrophotometer is a device used to measure absorbance by passing light
with a specific wavelength on a glass or quartz object called a cuvette. Part of the
light will be absorbed and the rest will be passed. The absorbance value of the light
passed will be proportional to the concentration of the solvent in the cuvette. The
working principle of the spectrophotometer is that the polychromatic light from the
light source enters the monochromator and experiences decomposition into
monochromatic light. The light is then transmitted through the cell containing the
sample. Light is partly absorbed by cells and partly transmitted to photocells which
function to convert light energy into electrical energy. The electrical energy
produced by photocells gives a signal to the detector which is then converted to the
absorption or transmittance value of the substance being analyzed (Khopkar, 2003)
The value of the absorbance results obtained in this practice is urea (0.203),
Compost (0.14), KNO3- (0.16), after making calculations with the formula y =
0.0651x + 0.0854, where y is the absorbance value and x is the nitrite concentration
then the nitrite concentration values obtained are Urea (1.80), Compost (0.83) and at
KNO3- (1.28). according to Hanafiah (2007), Giving KNO 3- is more effective
compared to urea or compost because KNO 3- contains elements needed by plants.
Urea has elements that plants need, but urea is acidifying the soil, whereas compost
does not carry the nutrients needed by plants. KNO 3 - should have a higher nitrite
content than the others (Harworth, 2015).
 V. CONCLUSION

Based on the result and discussion can conclude that: urea is more effective in
comparison with KNO3- or compos because urea has the highest absorbance
value and nitrate levels of 0.203 and 1.80. Effective administrasion because it
contains nutrients neede by plats.
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