Hypotheses Controlled environment Thuricin 17 enhances canola seed germination under low temperature, drought and the combination of high temperature and drought stress. Thuricin 17 promotes plant growth under low temperature, drought and the combination of high temperature and drought stress. Field conditions Thuricin 17 increases canola yield and oil quality under stressful conditions.
The research will be conducted in three phases: germination, plant development and a, under controlled environment conditions (growth chamber and greenhouse), and in the field. Production of thuricin 17 Extraction and purification Th17 will be carried out according Gray, et al., (2006a,b). Thuricin 17 will be produced by culturing Bacillus thuringiensis NEB17 in King’s B medium on an orbital shaker (Model 5430 Table Top Orbital Shaker, Forma Scientific Inc, Mariolta, OH, USA) at 150 rev min-1 for 48 h at 28 C.
Initial broth inoculum will be taken from plated material and grown in 250 mL flasks containing 50 mL of medium; a 5 mL subculture will be added to 2 L of broth in 4 L flasks under the same conditions as the initial culture for 96 h. Bacterial populations will be determined spectrophotometrically using an Ultrospec 4050 Pro UV/Visible spectrophotometer (LKB, Cambridge UK) at 600 nm.
A cell-free supernatant containing thuricin 17 will be prepared by centrifuging the bacterial culture at 13,000 —g for 10 minutes on a Sorvall Biofugre Pico (Mandel Scientific, Guelph, ON, Canada) and Th17 will be detected via analytical HPLC on a Vydac C18 reversed-phase column (0.
46 — 25 cm, 5 umol L-1). The HPLC will be fitted with Waters 1525 Binary HPLC pump and a Waters 2487 Dual ? Absorbance detector set (Waters Corporation, Milford, MA, USA) at 214 nm. Th17 will be isolated and purified by phase partitioning 2 L of bacterial culture against 0.8 L butanol for 12 h. The upper butanol layer will be collected by rotary evaporation (Yamota RE500, Yamato, San Fransisco, CA, USA) at 50 C under vacuum. Following evaporation, the extract will be resuspsended in 25 mL of 18 % acetonitrile (can: H2O, v/v) and then centrifuged at 13,000 —g for 10 minutes prior to HPLC analysis, as described above. Fractionation chromatography will be conducted under the following conditions: 45 min at 18 % acetonitrile, 45″110 min at 18″60.4 % acetonitrile, 110″115 min at 60.7″100 % acetonitrile and 115″120 min at 100″18 % acetonitrile. Th17 elutes in c. 60 % acetonitrile and is considered partially purified at this stage. Th17 will be stored at 4 C until further use, at which time it will be dissolved in HPLC grade water to produce uniform stock solutions which will be diluted to the desired concentration for each experimental treatment.
The experiment will be organized following a factorial where the two factors are stress level and thuricin 17 levels. There will be five replications of each experimental treatment. Every experiment described below will be repeated three times and the data are pooled for analysis. To disinfest the seed surface before treatment, seeds were soaked in 20 % bleach (6 % sodium hypochlorite, NaOCl) and agitated and rinsed with distilled water. I will be conducting the experiment in two Phases: in Phase 1, I will establish which concentration of thuricin 17( 10-7, 10-9, 10-11 M) enhances B. napus germination at a series of stressful temperatures (5, 10, 15, 30 0c) and optimal(25 0c ). In Phase 2, we will test the effect of thuricin 17( 10-7, 10-9, 10-11 M) solution under the combination of drought stress and various temperatures (5, 10, 15, 25, 30 0c). The osmotically induced drought (polyethylene glycol 8000 – PEG) levels will be control, -0.25 MPa (half strength Hoagland solution), -0.5 MPa (250 g L-1 PEG), -0.7 MPa (400 g L-1 PEG) or -1.3 MPa (550 g L-1 PEG 8000).
The growth chamber (Conviron) conditions for germination will be set to zero illumination, 65-70% humidity and designed temperatures. The experiments will be conducted in factorial completely randomized design with five replications and germination level will be assessed every 6 hours. Germination data were analyzed with PROC GLIMMIX in SAS 9.2 (SAS Institute Inc., Cary, NC, USA) with the binomial distribution option.Greenhouse work Plants will be grown in 10 cm pots and watered regularly with half-strength Hoagland nutrient solution. The growth chamber (Conviron) conditions for canola will be set to photosynthetic irradiance of 350-400 јE m-2 s-1, 65-70% humidity, 14/10 h (light/dark) regime. Experiments will be organized following a factorial with five replicates. The work will assess the utility of thuricin 17 in improving canola growth in the presence of three stress conditions:
I will not address high temperature alone in this work as we have just published key finding regarding this effect (Schwinghamer et al., 2015a,b). The two most effective concentrations from germination tests will be applied. Canola plants will be grown in pots to the three-leaf stage, and then be exposed to four levels of the stress in question, where one level is the control. The thuricin 17 will be applied in two ways, as a pre-planting seed treatment and as a spray at the time of stress. The temperature levels will be 5, 10, 15oC while the control temperature will be 25 oC. The osmotically induced drought (polyethylene glycol 8000 – PEG) levels will be control -0.25 MPa (half strength Hoagland solution), -0.5 MPa (250 g L-1 PEG), -0.7 MPa (400 g L-1 PEG) or -1.3 MPa (550 g L-1 PEG 8000).
In the case of heat and drought the same levels of drought stress will be applied, but at 30 oC. In each case, the plants will be allowed to grow for 2 weeks following the onset of treatment, and then harvested. The following data will be collected for growth and development at the time of appearance of first leaf: leaf area, height, dry weight, root branching, root fractal dimension, root length and root weight (leaf area with LI-COR leaf area meter and root variables with WinRhizo equipment). This information will be collected at the time of treatment imposition and at plant harvest, 2 weeks later. For plant physiology photosynthetic CO2 uptake rate, transpiration rate, stomatal conductance and CO2 concentration inside the leaves will be measured (using LI-COR 6400 portable photosynthesis meter, LI-COR Lincoln, NE); readings will be taken one day before treatment onset, one day after, one week after and just prior to harvest. Photosynthetic measurements will be taken on the upper-most fully expanded leaf of each plant, between 10:00 and 14:00 h. Chlorophyll fluorescence measurements (LI-COR 6400XT) will also be taken, at the same times as the photosynthetic measurements.
Plants harvested at the end of the experiment will also be used for final determination of elemental composition. Data analysis will be conducted using SAS 9.3 and differences between control and treatments will be considered statistically significant at P < 0.05, using Tukey's Honestly Significant Differences (HSD) test. Field work Canola will be planted each year on a range of soil types and with both early and late plantings. The variable soil types increase the likelihood of a range of moisture stress levels and the planting dates increase the likelihood of a range of temperature stresses. Planting dates will be early (late April) and normal (mid-May). Canola will be grown on three soil types, light (sandy), medium (loamy) and heavy (clay). The treatments will be organized on the field sites following a randomized complete block design with four complete blocks. Field plots will be 4 m long and the row spacing will be 20 cm, the seeding rate will be 5 kg ha-1. The sites will be fall tilled and secondary tillage will be carried out prior to seeding.
Soil samples (to 15 cm), will be collected prior to seeding and post-harvest for the determination of physio-chemical and soil-microbiome characteristics. Fertilization will follow standard agronomic practices, based on soil test results. Weed control will be performed manually. Field plots will not be irrigated. During the course of the season five plants per crop will be sampled at four phenological stages (mid-vegetative, mid-flowering, mid-grain filling, and harvest) for height, leaf area and dry weight determinations. At the end of the season 10 randomly selected plants will be removed from each plot, and assessed for yield components (eg.100-seed weight, number of seeds per reproductive structure, number of reproductive structures per plant), prior to machine harvesting of entire plots for determination of final seed yield. Statistical analyses will be conducted using Proc Mixed procedure in the SAS Statistical Package 9.3.Biochemical Analysis of seedsFatty acid compositionFatty acid composition, including saturated (sum of palmitic (C16:0), and stearic acid (C18:0) and unsaturated (sum of oleic (C18:1), linoleic (C18:2) and linolenic acid (C18:3) acids, will be analysed using gas chromatography of methyl esters following AOCS Official Method ce 1j-07.Iodine valueIodine value (iodine number) is referred to the total amount of unsaturation in fatty acids.
By definition, it is the measure of the number of grams of iodine, which will combine with 100 grams of the oil. Iodine value is a measurement of unsaturation calculated from the fatty acid composition according to AOCS Official Method cd 1c-85.Free fatty acidsBy an acid-base titration of the oil extracted from the seed during an oil content determination free fatty acids will be specified. Free fatty acid content will be determined by percentage of weight of oleic acid in the oil. The indicator and reagent options are listed in AOCS Official Method 5a-40.Glucosinolate contentGlucosinolates are ionic and water soluable determining by first extracting them from the seed into boiling water, and then isolating them from interfering components by ion-exchange chromatography Following the ISO 9167-3 method.
The oil content will be measured directly by grinding the seed and extracting the oil by AOCS Official Method Am 2-93. The oil content will be defined as the total of the substances extracted under operating condition in this method by percentage.Protein contentProteins are pivotal nutrients since they are a building blocks of tissues and key to metabolism. Crude protein is defined to describe the protein content of grain product or food, and protein measurement is done by determining it nitrogen content . The nitrogen content is then multiplied to 6.25 for oilseeds, to obtain the crude protein content. The Protein content will be determined by the AOCS Official Method Ba 4d-9o, revised in 2017.ProteomicsRoots and leaves at the vegetative stage and seeds at the harvest stage will be sampled for proteomics. After releasing protein from sample by using a protein extraction kit (Cat. no. PE-0230, Plant total protein extraction kit, Sigma-Aldrich, Co., St. Louis, MO, USA). The samples will be sent to the Institut de recherches cliniques de Montr©al (IRCM) for proteomic analysis using LC-MS/MS (Subramanian et al., 2016).
Protein profiling and IdentificationThe total protein extracts will be digested with trypsin and subjected to LC-MS/MS using LTQ-Velos Orbitrap (Thermo Fisher, MA, USA). Tandem mass spectra will be extracted, charge state deconvoluted and deisotoped, and all MS/MS samples will be analyzed using Mascot software (Matrix Science, London, UK; version 2.3.02). Mascot software will be used to search the canola databases assuming the digestion enzyme trypsin. Mascot will be searched with a fragment ion mass tolerance of 0.60 Da and a parent ion tolerance of 15 ppm. Carbamidomethyl of cysteine is specified in Mascot as a fixed modification. Oxidation of methionine is specified in Mascot as a variable modification (Subramanian et al., 2016). To validate MS/MS based peptide and protein identifications Scaffold will be used based on protein abundance in the samples.Contributions to knowledge:
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