Chapter 3: Materials and Methods
3.4 Modes of cultivation
3.4.3 Open Top Chambers (OTC) experimental site and
3.4.3 Open Top Chambers (OTC) experimental site and growing conditions
hrs per day. Eight fluorescent UV-313 (Q-Panel Company, Cleveland, OH, USA) with UV-B radiation of 280 and 320 nm emission was used to apply UV-B radiation treatments at the current (control) and elevated (UV-B) levels. The UV-B energy was installed about 1 meter above the plant canopy during the middle portion of the day and was checked daily at periodic time intervals. The UV-B lamps were directly mounted above each set of the plants receiving 8 hours of the UV-B radiation. The OTC units will precisely monitor temperatures and CO2 levels at fixed set points, as well as radiation levels that are similar to ambient.
Figure 3.11: OTC system diagram of the existing system in UAEU
At least three levels (optimum, medium stress and severe stress) of each stress factor were subjected under ambient (400 ppm) and twice the ambient level (elevated, 800 ppm) of CO2 concentrations during the different growth periods. Moreover, at least
two levels of each combined stress factor were also investigated under ambient and elevated CO2 and UV-B levels. The experimental treatment setup includes factorial combinations of two or more environmental variables in a completely randomized design.
The fully automatic control and monitoring system is the first of its kind in the UAE, which includes a CO2 analyser, UV-B monitor, PLC, and SCADA program with a PC to maintain the desired level of CO2 and UV-B within the OTCs. UV-B radiation was controlled by a dedicated computer system supported by a regulation of inlet valves (Uprety, 1998; Vanaja et al., 2006). The program allows you to set different CO2 concentrations in different OTCs at the same time, as well as adjust the time it takes to analyze the air samples taken from each OTC. The actual CO2 concentration and enhanced UV-B levels of each OTC are continuously registered and displayed in this facility. More details of operation and control of OTC chambers have been described by Karthishwaran et al. (2020).
Four pots of each type of water treatment with two wheat varieties and two replicates combined to a total of twelve pots on the 8th of January 2019.
Prior to planting, all pots were fertilized with manure at a rate of 30 t/ha.
Chemical fertilizers were applied once two days prior to sowing day at a rate of 120 kg P2O5/ha and 80 kg K2O/ha except Urea (Nitrogen) at a rate of 200 kg N/ha was applied 4 weeks after sowing day. Manual irrigation was 500 mL per pot per day (Figure 3.12).
On the 22nd of February 2020, approximately all 15 cm wheat plants were transplanted carefully from their pots to keep the soil around the new roots and to avoid
disturbing the delicate new growth to the OTC tubes (Figure 3.13). Manual irrigation was 200 mL per tube per day.
Figure 3.12: Transplanting of wheat Figure 3.13: Tubes for wheat plant in the OTC experiment
On March 21, 2020, wheat plant samples were collected to investigate differences in agronomy/physiological growth parameters such as plant and root length, plant weight and dry weight, Flag length and area, number of heads per plant, and head length. To avoid usage of CO2 and UV-B unnecessarily (harvest can happen after 4 months), the wheat plant was monitored for only one month after transplanting.
3.4.3.1 Analysis of morphological parameters
Plants were harvested 30 days after the UV-B, and CO2 treatments and the length of root was determined immediately followed by the measurements of plant height and number of heads. Plant height was measured in centimeters from the soil level to the tip of the shoot. The length of the plant roots was measured in cm from the
first cotyledonary node to the tip of the longest root. The total number of fully formed heads on each plant was counted and represented as a number of heads per plant. The leaf tissue constituents were measured in the fresh leaf material after 120 days after UV-B treatments.
3.4.3.2 Photosynthetic pigments Chlorophylls and carotenoid content
Complete phytochemical profiling of a representative plant sample is required before it is used for yield characteristics. Usually, the first step in the research with any crop is the profiling of their phytoconstituents. Chlorophyll, carotenoids, pigments which are generally secondary metabolites in a plant are known to affect the yield characteristics. The contents of chlorophyll and carotenoids were extracted from the leaves and calculated using Arnon's method (1949).
Extraction
In a pestle and mortar, 500 mg of fresh leaf material was pulverized with 10 mL of 80 percent acetone at 4°C and centrifuged at 2,500 g for 10 minutes at 4°C. The residue was removed again with 80 percent acetone until the green color was no longer evident.
The extracts were mixed and transferred to a graduated tube, which was then filled to a capacity of 20 mL with 80 percent acetone and immediately analyzed.
Estimation
Three milliliters of the extract were transferred to a cuvette and the absorbance was read at 645, 663 and 480 nm in a Spectrophotometer (U-2001–Hitachi) against 80
percent acetone as a blank. Chlorophyll content was calculated using the formula of Arnon (1949).
Carotenoid content was calculated using Kirk and Allen (1965) formula and expressed in milligram per gram fresh weight.
3.4.3.3 Enzyme extraction
The extraction procedure altered from that described by Samantary (2002) and Monnet et al. (2006). After each stage of the treatment, protocorms from each replicate were immediately removed. They were homogenized in 10 mL 100 mM potassium phosphate extraction buffer (pH 7.8) containing 2 mM EDTA ferric sodium salt and 2% (w/v) polyvinylpyrrolidone (PVP; R&M Chemicals, UK) using a precooled mortar and pestle (4°C). The homogenized centrifuged at 9000 rpm for 30 minutes. The supernatant aliquoted into 1.5 mL microcentrifuge tubes (MCT-150-C Microtubes, Axygen Inc., USA) and stored at -41°C (Sanyo Biomedical Freezer, MDF-U5411, Japan), for later use in the Bradford, antioxidant assays.
3.4.3.4 Estimation of soluble protein content
The soluble protein content of the protein estimated according to the method of Bradford (1976).
Extraction
20 mL of 20% trichloroacetic acid, one gram of fresh plant material, ground in mortar and pestle (TCA). The homogenate was centrifuged at 8000 rpm for 15 minutes.
The supernatant was discarded, and 5 mL of 0.1 N NaOH was applied to the pellet to solubilize the protein, and the solution was centrifuged for 15 minutes at 800 rpm. The
supernatant saved and made up to 10 mL with 0.1 N NaOH and used to estimate protein content.
Estimation
The Bradford assay was used to perform a quantitative measurement of protein concentration by preparing a typical protein concentration curve (Bradford, 1976;
Bollag et al., 1996). Samples with identified protein concentrations were tested alongside samples with unknown protein concentrations. A 100 µg.mL-1 bovine serum albumin (BSA, Sigma, USA) stock prepared by diluting 100 µg of the crystalline powder in 1 mL of distilled deionized water. Known concentrations of protein solutions prepared using the BSA stock diluted in the plant extraction buffer (Section 3.4) composed of 2 mM EDTA ferric sodium salt (R&M Chemicals, UK) and 2% (w/v) PVP in a 100 mM potassium phosphate extraction buffer (pH 7.8, Table 3.2).
Table 3.2: The contents of the Bradford assay reaction used in the generation of the standard curve
Standard protein concentration (µg.mL-1)
Amount of protein
(µg)
Standard solution (µl, from 100 µg.mL-1 BSA
stock)
Plant extraction buffer volume
(µL)
Bradford reagent (mL)
Blank 0 0 100.0 1.0
2.5 2.5 2.5 97.5 1.0
5.0 5.0 5.0 95.0 1.0
7.5 7.5 7.5 92.5 1.0
10.0 10.0 10.0 90.0 1.0
12.5 12.5 12.5 87.5 1.0
15.0 15.0 15.0 85.0 1.0
17.5 17.5 17.5 82.5 1.0
20.0 20.0 20.0 80.0 1.0
25.0 25.0 25.0 75.0 1.0
30.0 30.0 30.0 70.0 1.0
35.0 35.0 35.0 65.0 1.0
40.0 40.0 40.0 60.0 1.0
45.0 45.0 45.0 55.0 1.0
50.0 50.0 50.0 50.0 1.0
The assay was carried out at room temperature in plastic-capped culture vials (21 mm×85 mm) by diluting the necessary volume of BSA stock in the corresponding volume of extraction buffer, with both totaling 100 µL in volume. The Bradford reagent (1 mL) was then applied to the mixture. The mixture was vortexed for 10 seconds, then allowed to sit for two minutes before being poured into 1.5 mL plastic cuvettes and measured at 595 nm with a spectrophotometer. For the PLB samples, 100 µL of each extract aliquoted into 1 mL of the Bradford reagent, vortexed for 10 seconds, allowed to stand for two minutes, poured into 1.5 mL plastic cuvettes and read in a spectrophotometer at 595 nm. Any samples are presenting with optical densities (OD) that were outside of the assay’s linear range of from 0.2 to 0.8 OD units repeated with the mentioned dilution factor, if any.
3.4.3.5 Estimation of total free amino acid content
Moore and Stein (1948) method was used to extract and estimate total free amino acids.
Extraction
In a mortar and pestle, 500 milligrams of fresh plant material were homogenized with 10 milliliters of 80 percent boiled ethanol. The extract was centrifuged for 15 minutes at 800 g, and the supernatant was made up to 10 mL with 80 percent ethanol and used for the calculation.
Estimation
In 25 mL test tube, one milliliter of ethanol extract was taken and neutralized with 0.1 N NaOH using methyl red indicator. To which, 1 mL of ninhydrin reagent was added. The content was boiled in a boiling water bath for 20 minutes, and then 5 mL of
diluting solution was added, cooled and made up to 25 mL with distilled water. A Spectrophotometer (U-2001–Hitachi) was used to measure the absorbance at 570 nm against a suitable blank. The amino acid content was measured using the standard graph, which was prepared using Leucine as a standard, and the results were expressed in milligram per gram dry weight.
Reagent
Ninhydrin Reagent
Solution I: 80 mg of stannous chloride in 50 mL citrate buffer at pH 5.0.
Solution II: 2 grams of Ninhydrin in 50 mL methyl cellosolve, both solutions were mixed freshly.
Diluting Reagent
Distilled water and n-propanol mixed in equal volume (1:1 v/v).
3.4.3.6 Total phenols
The total phenols were calculated using Nagarajrao et al. (1980) method. In the Folin-Ciocalteau reagent, phenols combine with phosphomolybdic acid to create a blue- colored complex in an alkaline medium that can be measured spectrophotometrically at 660 nm.
Reagents
1. Folin-Ciocalteu Phenols 2. Absolute ethanol.
3. Standard catechol
4. 2% (w/v) sodium carbonate.
The sample (0.5 g) was homogenized in 10X volume of 80% ethanol. The homogenate was centrifuged at 10,000 rpm for 20 minutes. The extraction was carried out once more with 80% ethanol. The supernatants were collected and evaporated until they were fully dry. The residue was then dissolved in distilled water in a known amount. Different aliquots were pipette out, and purified water was used to fill each tube to 3.0 mL. Folin-Ciocalteau (0.5 mL) and 2 mL 2% (w/v) sodium carbonate were added, and the tubes were placed in a boiling water bath for precisely one minute. The tubes were cooled, and absorbance was read at 660 nm in a spectrophotometer against a reagent blank. Standard catechol solutions of different concentrations were also treated as above, and the standard curve was prepared. The concentrations of phenols were expressed as mg/g of fresh tissue. Total phenols = ((Abs+0.019)/1.17) x (volume/weight)
3.4.3.7 Enzymatic Antioxidants
3.4.3.7.1 Assay of superoxide dismutase (SOD, EC 1.15.1.1)
Superoxide dismutase in the plant sample was examined by the method of Hwang et al. (1999). The assay is based on inhibition of the formation of NADH- phenazine methosulphate, nitroblue tetrazolium formazan.
Reagents
1. Sodium pyrophosphate buffer: 0.025 M, pH 8.3.
2. Absolute ethanol.
3. 1.3 µM riboflavin
4. Phenazine methosulphate (PMS): 186 mol.
5. Nitroblue tetrazolium (NBT): 300 mol.
Extraction
10 mL of ice-cold 50 mM sodium phosphate buffer containing 1 mM PMSF was used to homogenize one gram of fresh tissue. A double-layered cheesecloth filter was used to filter the extract. At 4°C, the extract was centrifuged for 20 minutes at 12,500 rpm. The supernatant was saved and used to estimate SOD enzyme activity by diluting it to 10 mL with extraction buffer (Table 3.3).
Estimation
The activity of superoxide dismutase was calculated as defined by Beauchamp and Fridovish (1971). The reaction medium was prepared, and 1 mL of enzyme extract was added to 3 mL of reaction medium. The reaction mixture contained 1.17 x 10-6 M riboflavin, 0.1 M methionine, 2x10-5 potassium cyanide and 5.6 x 10-5 M nitroblue tetrasodium salt (NBT), dissolved in 0.05 M sodium phosphate buffer (pH 7.8). Two sets of Philips 40 W fluorescent tubes were used to light the mixture in glass test tubes. At 30°C for one hour, illumination began to initiate the reaction. Those that were not illuminated were saved as blanks and held in the dark. The absorbance was read at 560 nm in the spectrophotometer against blank. The function of superoxide dismutase was measured in units. Under the assay conditions, one unit is defined as the amount of change in absorbance by 0.1 per hour per milligram protein (Cherry, 1963).
Table 3.3: Composition of sodium phosphate buffer (pH 6.1) 0.1 M
Compound Quantity
Na2HPO4.2H2O 35.61 g/L
NaH2PO4.H2O 27.6 g/L
ddH2O Top up to 1 Litre
A total of 467.5 mL of NaH2PO4.H2O was mixed with 92 mL of Na2HPO4.2H2O. Then pH was adjusted with NaH2PO4.H2O until it reaches 6.1.
3.4.3.7.2 Peroxidase activity (POX, EC 1.11.1.7)
Kumar and Khan's procedure was used to calculate peroxidase (1982). 2 mL of 0.1 M phosphate buffer (pH 6.8), 1 mL of 0.01 M pyrogallol, 1 mL of 0.005 M H2O2, and 0.5 mL of enzyme extract made up the Peroxidase assay mixture. After a 5-minute incubation time at 25°C, the reaction was stopped by adding 1 mL of 2.5 N H2SO4.By measuring the absorbance at 420 nm against a blank prepared by adding the extract after 2.5 N H2SO4 at zero time, the amount of purpurogallin produced was calculated.
The behavior was calculated in units of mg-1 protein, with one unit equaling 0.1 min-1 mg-1 protein shift in absorbance.
The peroxidase enzyme activity calculated as specific enzyme activity where the one unit of enzyme activity is the amount of enzyme used to reduce hydrogen peroxide in the reaction vessel.
The formulas used to calculate the specific enzyme activity of peroxidase are as follows:
Absorbance value x df x 1000 Total peroxidase activities =⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯
Sample
Where df = dilution factor and sample = amount of supernatant used to react with H2O2
in mL
Total peroxidase activities
Specific Enzyme Activity (U /mg) =⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯
Soluble protein content in mg 3.4.3.8 Glutathione reductase
Griffith's approach (1980) was used to quantify reduced glutathione (GSH) in plant tissues. When 5, 5′-dithio-bis (2-nitrobenzoic acid) (DTNB) was applied to compounds containing sulphydryl groups, 2-nitro-5-thiobenzoic acid (a yellow-colored compound) was formed.
Extraction
In a pestle and mortar, 200 milligrams of plant material were ground with 5 milliliters of 2% metaphosphoric acid. The extract was centrifuged at 17,000 g for 10 minutes, with the supernatants saved for examination.
Estimation
To 0.9 mL of the extract, 0.6 mL of 10 percent sodium citrate buffer was added for neutralized the extract. The assay mixture 1 mL containing 700 μ1 NADH (0.3 mM/L), 100 μ1 Dithionitrobenzoic acids (DTNB) (6.0 mM/L), 100 μ1 distilled water and 100 μ1 of neutralized extract. This mixture was stabilized at 25°C for 4 minutes, and 10 μl of Glutathione Reductase (sigma) was added to this and read at 412 nm against appropriate blank.
Preparation of Reagent
Glutathione Reductase (sigma VI type) obtained from Sigma-Aldrich, USA.
3.4.3.9 Estimation of catalase (CAT, EC: 1.11.1.6)
Catalase activity was assayed as described by Chandlee and Scandalios (1984) with modification.
Reagents
1. Phosphate buffer: 50 mM, pH 7.0 2. Hydrogen peroxide: 15 mM
3. Phenazine methosulphate (PMS): 1 mM 4. Standard hydrogen peroxide: 1 mM Extraction
In 5 mL of ice-cold 50 mM sodium phosphate buffer (pH 7.5) containing 1 mM PMSF, 500 mg of frozen content was homogenized. The extract was centrifuged for 20 minutes at 12,500 rpm at 4°C. The enzyme assay was performed on the supernatant.
Bradford (1976) procedure was used to assess the enzyme protein.
Assay
The activity of the enzyme catalase was calculated using a modified Chandlee and Scandalios (1984) process. 2.6 mL of 50 mM potassium phosphate buffer (pH 7.0), 0.4 mL, 15 mM H2O2, and 0.04 mL of enzyme extract made up the assay mixture. The drop in absorbance at 240 nm coincided with the decomposition of H2O2. 1 mM H2O2
reduction per minute per mg protein was used to calculate enzyme activity.
3.4.3.10 Proline oxidase activity (L- Proline: O2 Oxidoreductase, EC: 1.4.3.1) Proline oxidase activity was determined according to the method outlined by Huang and Cavalieri (1979).
Extraction
One gram of plant tissue was taken in a pre-chilled mortar and pestle and homogenized with 5 mL of homogenizing medium. The filtrate was centrifuged at 10,000 g for 10 minutes in a refrigerated centrifuge at 4C. The supernatant was again recentrifuged at 20,000 g for 25 minutes. After completion of fractionation, a pellet was obtained. This pellet was mixed with 1 mL of 5 mM Tricine – KOH buffer (pH 7.5) containing 6 M sucrose. This extract was used for assaying the enzyme activity. The extraction was carried out at 4C.
Enzyme Assay
The enzyme was assayed spectrophotometrically. Three milliliters of assay mixture contained 0.1 mL of enzyme extract, 1.2 mL of 50 mM Tris-HCl buffer at pH 8.5, 1.2 mL of 5 mM MgCl2, 0.1 mL of 0.5 mM NADP, 0.1 mL of 1 mM potassium cyanide (KCN), 0.1 mL of 1 mM phenazine methosulfate (PMS), 0.1 mL of 0.06 mM 2, 6-dichlorophenol indophenol (DCPIP) and 0.1 mL of 0.1 M proline. Proline was used to start the reaction, which was controlled at 600 nm at 25°C. It was noted that the optical density increased. The enzyme activity was determined by the rate of DCPIP reduction. The activity of the enzyme was calculated in millimoles of DCPIP reduced per minute per mg of protein.
Homogenizing Medium
50 mM Tris-HCl buffer (pH 8.5) 0.06 M sucrose
50 mM Tricine-KOH buffer (pH 7.5) Proline Metabolizing Enzymes
3.4.3.11 γ - Glutamyl kinase activity
[ATP: L. Glutamate 5- phosphotransferase (EC: 2.7.2.11)]
γ-Glutamyl kinase activity was assayed by the method of Hayzer and Leisinger (1980).
Extraction
In a vortex homogenizer, one gram of plant tissue was extracted for three minutes with 10 mL of 50 mM Tris-HCl buffer (pH 7.2). The cells were then centrifuged for 20 minutes at 10,000 g, washed with 50 mM Tris-HCl buffer (pH 7.2), and deposited at –20°C. 1 mM 1, 4-dithiothreitol was applied to a frozen sample suspended in 7 mL of 50 mM Tris–HCl buffer (pH 7.2). A double passage through a French press at 38.5 MPa caused cellular damage, and the cell debris was removed by centrifugation at 20,000 g for 30 minutes. The crude extract was saved and used to measure γ-glutamyl kinase activity. All operations were carried out at a temperature of 4°C.
Enzyme Assay
Crude enzyme extract (2.5 mL) was desalted with a Pharmacia PD-10 (SephadaxG-25) column equilibrated with 50 mM Tri-HCl buffer (pH 7.2), containing
1 mM 1, 4-dithiothreitol. The enzyme assay mixture contained, in a final volume of 0.25 mL; L-glutamate, 50 mM; ATP, 10 mM; MgCl2, 20 mM; Hydroxylamine HCl, 100 mM; Tris base 50 mM (pH 7.0) and 100 l of desalted extract containing approximately 3.0 mg enzyme protein in a final volume of 2 mL. After 30 minutes of incubation at 37°C, 1 mL of a solution containing FeCl3.3H2O (2.5% w/v) and trichloroacetic acid (6% w/v) in 2.5 M HCl was added to start the reaction. Precipitated protein was removed by centrifugation at 10,000 g (0C) and the absorbance was measured at 535 nm in spectrophotometer against a blank (water). One unit of - glutamyl kinase activity can be defined as g of -glutamyl hydroxamate formed per minute per mg protein. -Glutamyl hydroxamate was used as standard.
3.4.3.12 Statistical analysis
To assess the effect of UV-B and CO2, the variance analysis (ANOVA) was performed using SPSS software (v. 21.0). Duncan's multiple range test was used to assess the substantial difference between means at the P≤0.05 stage. The SAS software's (SAS 9.4) PROC PRINCOMP method was used to conduct principal component analysis (PCA). PCA is a multivariate statistical tool used to separate experimental units (UV-B and CO2 treatments) into subgroups based on the measured parameters (e.g., growth and leaf constituents) by producing the loadings of these parameters (termed as eigenvector) and principal component scores for each unit. P- values were taken as an indicator of significant differences among the treatments.
Chapter 4: Physiochemical, Elemental, and Bacteriological Analysis