Effects of nitrogen on the activity of antioxidant

enzymes and gene expression in leaves of

Populus plants subjected to cadmium stress
Abstract:

Research aims to confirm the main physical effect of nitrogen (n) on the codium (CD)
plants. Thus the plants were enhanced in the nutritional solution of Hoganland and was
treated with additional N, CD, and N + CDs. After the treatment, plant growth and
chlorophilic material was recorded. Oxident stress, anti-oxidation activity, and related genes
were also expressed. The results indicated that the cured cereals with single CD have given
clear toxic symptoms, such as development exposure, depositing oxygen species, and lack of
chlorophilic materials. However, when CD was added to stress under stress, plant growth was
increased, chlorophilic synthesis was promoted, and oxidatic stress was eliminated. In
addition, antioxidant enzymes are expressed by Anne. The results have been pointed out that
N has denied the toxic effect of the CD on partially populated plants, which can provide a
new procedure to increase the futurization technology for heavy metal pollution.

Human development, such as industrial and urbanization, have created serious

environmental pollution problems. The increasing amount of heavy metals in the
environment makes a serious threat to the natural ecosystem, causing its discomfort to
worsen. Cademium (CD) is one of the most toxic pollution found in air, water and soil, and it
is unnecessary for plants. It can be stored by food chain (Wagon 1993 Wagner G. 1993.
Cademium deposits and crop results result in human health results in recent crops. CD
pollution has become more intense and recently I have become a cause of disadvantage,
including the destruction of farmers. The world's primary pollution areas (Berne and E. 2012,
Berne, Fraig K, Sororurora P, Ferrora M., Nigro F, Cncc 2012. 2012) Collect and distribute
heavy metals and durable metals in flour and herbs, flourished under the conditions of
Romania.

CD tension is mainly encouraged to collect reactive oxygen species (ROS), which

include Super Oxide Radicals (O2 -), Hydroxyl Radicals (OH), and Hydrogen Peroxide
(H2O2) Oatts (atoms) or atoms of atoms are at least one unpaired electron, as free extremism,
O2-, OH, H-, CH3- Free-based oxygen free radicals in the plant (NR R). OfR has more than
95% free radicals in accounts and is mainly the result of the process of oxygen. These free
radicals can return to other molecules electronics, and can reduce the structure of DNA,
RNA, and protein to reduce plant body. Usually, ROS generation and endurance speed is
balanced due to the activity of protective enzymes, including Super Oxide Dropas (Sod),
Kittsast (CAT), Gutthane Peroxide (GPX), and Acurateate Peroxide (A PX) are included .
Chloroplast: Enzymol formation of active oxygen and its drilling methods. .. However, when
planting is planted during tense periods of time, then Maximum ROS ends. As soon as
balance is broken, due to the maximum accumulation of ROS in the plants, the Lip-Peroxide
reaction significant damage to the plant membrane system. This is, which may increase the
parliamentary and conductivity. Heavy metal ion affects the effect of sun flower leaves
aksaydytk evidence of the involvement of stress. Our first study shows that after the
treatment of n, the damage caused by CD stress can decrease. In addition, these plants are not
better than the plants. However, the result of this result is not clear, because the generation of
independent fields is not well studied under the plantation of CDs and non-motivated defense
mechanisms in the plants. Therefore, it is important to investigate the gene expression of N
and Anti-oxid anemens, such as SOD, CAT, GPX, and APX, will help eliminate free oxygen
bases during CD stress. In addition, we want to detect N-detoxification mechanisms of heavy
metal tension in the plant, which can provide new ideas and procedures to increase
phytoremediation technology for heavy metal pollution.

Cutting Chenor Plants '107' was chosen as a large range of experimental materials and
NH4NO3 and CdCl2 studies. Forty-five nodes and 15 cm height, forty-five healthy beds, as
well as 48 different trees of Populus deltoides × P-Nrara, were displayed in the south-west,
Ya'an. These trees were collected from the same population and similar water and soil
nutritional conditions were combined. After samples, these 48 balls were converted into 16
plastic tubes (3 cut per tube, every tube 100 × 50 × 20 cm, and maximum 60 L solution), and
every tube hogan land nutrition Was full of solutions. The balls were growing at a naturally
light lamp house in Sechon Agricultural University, and the solution to the land of Hlandland
was changed every 96 hours. When cuttings were 8-10 leaf phase (2-3 weeks, 20 cm height),
they were subject to the following four treatments: (T1) No CD and no extra n (control;
standard Hoagland nutrition solution); ( T2) Additional N (45 μM) and no CD, NH4NO3 of
2.10 g 1 L Hoagland nutrition solution (equivalent to 3 limit of standard Hoagland nutrition
solution 3); (T3) CD (50 μM) And no extra n, 0.0059 g CdCl2 1 L Hoagland nutrition
solution included; and (T4) CD (50 μM) and Extra N (45 μM). The quantity of CD (CD2 +)
was equal to every single container involved in each vessel (T3 and T4); Similarly, the
quantity of n per vessels was equal to T2 and T4. Each treatment includes 4 tubes and 12
pieces (table 1). Leaf samples were cultivated after 1, 6, 12, 24, 48, 120, 240, and 720 h, and
then frozen liquid enzymes for anemia activity and genetic expression. During 30 days of
treatment, treatment solution was changed to change every 48 h so that the same
concentration should be maintained.

The height of each cutting plant and DBH (diameter at the height of diameter) was
measured before and after treatment. From the bottom of the leaves, lef samples were taken
immediately from the bottom of the plants with a third leaf count and used to determine
chlorophel material. Chlorophyl was removed in 80% of acne. Emotional 663 NM and 645
NM were measured by a spectromolytometer (UV-2450, Shimmaz Corporation, and Japan).
Lichthalmel (1987 Lucknow H.H. 1987. Chlorophys and carotenidis: equality and equality of
the costs of photopensheit biomanians were used to calculate the quantity of angular
chlorophyll. Measurements were done in trials.
H2O2 and MDA concentration analysis
After treatment of 30 dye, H2O2 treatment was found in treatment and control plants found
by FOX1 (Bellincampi et al 2000 2000Bellincampi D, Dipierro N, Salvador G, Kroneon F.,
Lorenzo GD 2000. 2000. By Oligogalacturonides Encouraged Extracellular H2O2 is not
included in the entertainment of tobacco leaves in order to show impaired regulation roller B-
gen expression.). Fresh leaves (4 grams) were gayed with 8 mm of 100 mm sodium
phosphate buffer (PHO 7.2) and 10% (W / V) polyvinylopolopolodone (PVPP) (LLL). 2009
Lewis SS, Jones S., Campbell-Palmer L. 2009. A revised chemistry of determining hydrogen
peroxide in Apple Fruit tissues. Homogenates 4 4 cm 10,000g for 10 minutes in 10 minutes
was setrifuged. To determine the concentration of H2O2 concentration, 500 μL of
supernatant was included in 1 mL dental regent (500 μm ammonium ferrous sulfate, 50 mm
H2SO4, 200 μM xylenol orange, and 200 mm sorbitol and 200 mm. 100 mm 500 μl of Easter
Sodium phosphate Buffer (PHP 7.2). Records absorbed at 560 mm was recorded by a
spectrometer (UV-2450, Shimadzu Corporation, Japan) after integration after 30 minutes in
the dark. H2O2 standard Depending on every test experience, the H2O2 concentration (nmol
• G-1 • FW) was calculated by the difference between the emission of empty and the test
sample.

Total soluble protein analysis

Using the leaves, measuring total soluble proteins was smooth in 50 mm THHL, PHO
7.5. 0.04% (v / v) 2-mercaptoethanol and 2mm EDTA. Then, for 4 minutes, for 21 minutes,
11,500 grams were centragrams. Surfitant has been saved in 20-20 cc for analysis. The
concentration of protein was set according to Birdfield (1976 Badford MM) procedure. 1976.
The amount of protein micragram using the principle of protein dyeing is the fastest and
sensitive method of quantity.

Analyze inventory activities

Super Oxide Disclaimer (SDD)

For the rise of the SOD, the fresh leaves (0.5 grams each sample) were ground with
liquid n in pre-sliced pearls and peas and 50 mm extracted with potassium phosphate buffer
(PH 7.8), 0.5 mm MIDE was included. Each homogenate tubes were transferred corrosion
and was centrifuged at 15 ° C for 15 minutes, 4 ° C for 15 minutes. Spentant was used for
harmony activity. Total SOD activities were ensured by monitoring the exhibition of the
photographic deficit of Nightproof Teterasculum (NBT) as per seram and L modes. (2002
Serim R.K., Rao K.V., Savitsas G.C.2002. The differences of long-term proportion of
nutrient genitypes in relation to oxidative stress, anti-oxidation activity and heavenly
concentration. A unit of acid activity caused 50% of anemia The quantity of NBT was
described as a quantity of 560mm was monitored as a quantity.
Catalase (Kate) Activity

The origin of the CA was like extracting the SOD. The initial rate of CAT's anti-
activity activity H2O2 decreased (Abbey 1984Aebi H. 1984. The initial rate of Catalase in
Vitro was followed. 3 mm reaction mixture 100 mm PBS (Pho 7.0), 10 mm H2O2, and EGM
activity consisting of 100 μl of antioxidants extracted Enzyme activity by monitoring
absorption reduction on 240 mm resulting in the consumption. The capacity of extracting the
enzyme activity for H2O2 (0.036 mm -1) Cm-1). As per the amount of mic per H2O2 per
minute of the protein to erase 1 μmol per minute.

Oscerbet Per Peroxide (APX)

According to APASA (Osborne oxidation) oxidation, the spectrophotometer was

determined. Fresh leaves (0.5 grams each sample) were smooth in 50 mm Ice Cold Potassium
Phosphate Buffer (PH 7.5), with 0.5 mm SDD A, 2 MOSSurate (SS) and 5% Polylone
Perrolone (P) VP) was included. Pearls and mortars. Other measures were like extraction of
other enzymes

Guaiacol Peroxide (GPX)

The GPX activity was measured using the singular coil. GPX was pulled into NASC
and L method. According to 2.77 millimeters of 50 mm phosphate buffer (PHO 7.0) of 25 μl
of anesthesia extraction (PHO), eliminate oxidant loss in the underwater tomato plant in the
underwater tomato plant. Impact of unusual Argentina for. ), 0.1 mm 1% H2O2 (V / V), and
4% guaiacol (0.1 / G) 0.1 mm (V / V) 0.1 mm. Due to gyoyysol oxidation, 470 mm was
recorded for 3 minutes in absorption. One unit of angle activity was expressed per minute as
a result of the 0.01 change in absorption.

Anti-oxidant anthem gene expression

RNA isolation

The total was extracted from RNA leaves, as described by Vererader and L. (1989
Herododd TC, Decker BMM, Hukema A. A. 1989. Rule of RNA was certified by
electroprocesses using Ethiopian Bromide Stingong. First of all, CDA's motivated tech red
Scripts ™ were reverse according to RT-PCR kit's standard protocol. ΜL of 500 NRA, 1.0 10
mm ml / LDNTP, 1.0 μl of 2.5 μmol / μl OligodT primer, 20 μmol / μl random 1.0 μl of
primer, and 2 μl of RNase-free H2O was mixed and 65 ° C for 5 minutes. This mixture was
immediately placed on ice for 2 minutes and then a short time Center centurized for this After
5 × 4.0 μl of standby buffers, 0.5 μl of 40 y / μl prevention prevention, 0.5 μl of primer
scripts ™ RTase, and 5 μl RNase-free H2O were added and mixed. The volume of 20 μl was
stopped for 30 minutes, 45 minutes, 45 minutes, 5 minutes on 25 minutes, 5 minutes for 5
minutes, and warm for 15 ° 70 ° C to prevent analysis reaction in minutes And then held on 4
° C.

Cloning & Configuration

 Partial pieces of selected genes are available in clones and our laboratory (data is not
shown). Partial pieces layout figures were collected in Jane Beckinc, and the numbers listed
in Table 2 are listed.

Realtime RT-PCR

The first CDNA tips were attached to 500 NNN treated RNA as the following
statement. Genie based on related steps for Cu / Zn-SOD, CAT, GPX, and APX was designed
using special premises Press Express software, version 2.0 (uploaded bius system,
courtabout, france). Jenny express profiles of CD stress and an exhibition response were built
using real-time RT-PCR with specific premises (Table 2). Real-time quantity was performed
by PCR Bio Redi Q5 (bio-red company), and the analysis was analyzed, which is the Optical
System Version 2.0 (bio-rd) software. Thus the internal reference genre is used as a genus,
which allows minor and quantity to be allowed, with only target genes. Based on the reaction
of the SYBR premium premium ex-Talk ™ II (Figure) reaction protocol, the reaction system
was listed below: SYR Premix Ex Taq ™ II (10 ×) 10 μl, 0.8 μl of each primer (10 μmol / l),
1.5 μl template (cDNA solution), and 6.9 μl of dH2O. The two-step PCR reaction program
was presented as a process: 95 ° C 10, 95 ° C, 5 and 55 ° C 20, PCR reaction with 20 cycles,
and then at 72 ° Is kept For 2 minutes. After the PCR reaction, the end of each part of
determining the purification of a melting curry produced product for every durable. Real-time
PCR results were done in three times (Technical Re-) to ensure the re-creation of results. The
level of relatively express was used using the transmission of 2-ΔΔCT methods which was
calculated as the transmission level, such as Livak and Schmittgen (2001Livak KJ,
Schmittgen TD. 2001.) PC in real-time quantity RNA Jane expression express data using R
and 2-ΔΔCT method.

Statistical analysis

The figure is expressed as standard error, unless otherwise stated. All data was
analyzed by SAS Software (Version 8.0, SAS Institute, Carrie, North Carolina, USA). All
figures met with common equality and variable equality. Variable analysis (nova) was used to
determine the difference between later analysis and treatment groups. At least when required,
the minimum key comparison (LSD) test was used for comparing multiple. Differences in P
<0.01 were considered significantly important.

Results
Effect of CD treatment on plant development

30 days treatment is shown in popper height and DBBT table. The results show that in
the study period only leaves a major development show in the address of the plants with CDs.
Regarding average height and DHB, 42.3 percent and 29.8 percent respectively compared to
control. Under the sole control of contrast, the height and the DBB increased 43.3 percent and
35.4 percent respectively. In addition, the plants associated with the N + CD well without any
toxic symptoms, and an important increase in growth was seen in 30 days (regarding 70.3%
and 63.1% control) (Table 3). ). The difference in the development of the plants between the
treatment was very important, and the CD and N interaction significantly promoted Popar's
growth more than the n (comparatively tables 3 and 5).

Effect of CD on chlorophilic material

To evaluate oxidatic stress due to CD, chlorophil content was checked after 30 days
after treatment. Chlorophyll material differs widely in different treatments (Table 4). After 30
days of CD treatment, according to control values, respectively, Chl B, and total Chl, 30%,
48%, and 35% was lacking. Chlorophile was significantly higher than the ratio (4.62: 1)
control (3.43: 1), which indicates the severe exposure to the CD on CHL b composition.
Amazingly, chlorophile levels were increased in the populous cured with total + total. An
important increase of 69.6%, 41.2%, and Chl 63.2% was found in the day 30 (table 5)
compared to CHL, and total CHL control. The results show that chlorophile material can
significantly increase in leaves under CD stress.

Effect of CD on H2O2 and MDA Material

H2O2 and MDA membrane are liped-peroxide products, and their material is related
to the degree of membrane liped pipe oxidation. Thus, they work as a major indicator of
heavy metallurgy stress. The trends of H2O2 and MDA elements in all treatments were
shown in Figure 1. Thus the plants of the H2O2 and MDA increased by expanding the cdCl2,
and their content in cereals with CD only cure and control. Interestingly, their content has
shown a significant decline in the plants associated with the N + CD compared to the same
CD treatment, on the study period (single shape), while their material during the study period
Maintained at low level in the plants associated with the NH4NO3. The results have been
pointed out that in the tension of CD 2+, the stress of CDs promotes the accumulation of
oxygen species (ROS) and N supplement under the stress of CD + + reducing the production
of ROS.

Effect of CD on SOD, CAT, GPX, and APX activities

Anti-oxidant system is one of the protective mechanisms that can eliminate reaction
oxygen species (ROS) in plant cells and stress stress-inspired oxidic loss (Bakakk & Scholar's
2003 2003, Scanner DG. 2003. ) During the cold acclimation, the anti-oxidant digestion of
the expression of expression of oxygen wheat near the gene expression. As it can be seen in
Shape 2, the CD exhibit has left significant changes in the Sod, CAT, GPX and APX
activities in the study period in Popper. To control, but significantly increased under Cd and
N + CD treatment. In addition, SOD activities were stuck under the CD and N + CD stress at
24 H. Average activities for the SOD were in this order: N + Cd> Cd> N> CK (Figure 2a).

Result
There is a dangerous effect on the development and development of CD pollution
plants. The maximum concentration of CD in the plants shows different symptoms of toxic,
including exhibit in growth, cholorto leaves, and biomass deficiency. In this study,
researchers discovered that napper (C delotides × P.Nagra) can effectively reduce CD
frequency loss. Inserting Poplars Appendix N under CD stress can help increase the growth
of the plants, promote chlorophilic synthesis, and promote anti-oxidant enzymes and gene
expression activity. As a result, this indicates that partially reduced the toxic effect of the
metallurgy on the poplar plants, and could significantly contribute to the detoxification of
heavy metallurgy stress on the plant.

Additionally, it is notable that our experiences have only studied the effects of anti-
oxidation and gene expression of gene in the pope, and did not study further on other
metabolic paths, such as protein synthesis. As a result, under CD stress, our ideology is not
completed on the plant's detoxification effect and mechanism and further study is needed.

Recognized parts

This work was supported by the 12th year key programs for forest generation in the National
Natural Science Foundation (No. 31300514) and Sichuan Province (No. 2011YZGG).