Plant matter and soil
Snap bean (P. vulgaris. cv. ‘Bronco’) is a primary vegetable crop widely grown in the United States. The seeds of “Bronco” are commercially available for research and breeding and were purchased from a seed company, HOSS TOOLS located in Norman Park, GA (GPS coordinates: Latitude: 31.2462 and Longitude -83 .6549) before the start of the study in the spring of 2015. Miracle -Gro® garden soil was purchased from Lowes in Gainesville, FL.
In Trial 1, snap bean seeds were soaked in 0.15% H2O2 for 24 h and rinsed with deionized (DI) water 5 times, then placed in a 12 cm petri dish with moistened filter paper and sealed in a plastic zip-lock bag and incubated at 33 °C for the night. After seedling emergence, each seedling was transplanted into a 5.1 cm diameter plastic wire basket. The baskets (Net Pots Net Cups, Heavy Duty Plastic Net Pot with Wide Rim Design) have been placed in an aeroponic system to allow rapid root growth and development. The aeroponic system consisted of a tank half filled with 10% Hoagland solution26, a misting pump, tubing and three sprinklers that continuously mist the roots. After two weeks, seedlings with three true leaves were transferred from the aeroponic system to a hydroponic system where the roots were immersed in 1000 mL of 0.2 mM CaSO4 solution for a measure use. The use of CaSO 0.2 mM4 solution was to protect the membranes, because calcium is essential for the integrity and selectivity of biological membranes27.
In Trial 2, two pot sizes were used for different purposes. The small pots 7.6 cm in diameter × 8.9 cm in height were intended for seed germination. The largest pots of 15 cm in diameter × 16.5 cm in height for production. Miracle-Gro® garden soil was used and fertilizers were applied per pot as follows: NH4NO3, 2.92g; triple superphosphate, 1.65g; potassium chloride, 0.44 g per jar.
The trials were all conducted at the Department of Horticultural Sciences, University of Florida/IFAS, Gainesville, Florida. The hydroponic trials consisted of three sub-trials. Trials 1–1 and 1–2 had the same treatments with four replicates. Trial 1–1 was conducted in a controlled environment growth chamber with a temperature range of 23-27°C, 16 h daylight and 8 h darkness. Trial 1–2 and Trial 1–3 were conducted in a standard greenhouse with a temperature range of 23–27°C. Test 2 was carried out in a high tunnel under a natural atmosphere. Treatments were imposed by (1) flood control without bubbling or application of H2O2 after preset time (approximately 100 μM DO level at start, no supplemental oxygen), (2) aeration (continuous oxygen supply with an air pump), and (3) H2O2 (application of 529 μM H2O2 at 0h). The growth medium was 1 L of DI water with 0.22 g L−1 K.H.2Purchase order40.373 g L−1 TO KNOW30.02g L−1 CaSO4, 0.012gL−1 MgSO40.29mg L−1 H3BO30.18mg L−1 MnCl2∙4H2O, 0.02mg L−1 ZnSO4∙7H2O, 0.008 mg L−1 CuSO4∙5H2O, 0.002mg L−1 H2moo4∙H2O, 0.65mg L−1 Ethylenediaminetetraacetic acid (EDTA) and 0.62 mg L−1 FeSO4∙7H2O. For trial 1–1, 2 ml samples were taken from the center of the jars every 2 h for 10 h. Samples were analyzed for ortho-P (EPA method 365.3) and NO3− (EPA Method 352.1) using a Seal AQ-2 discrete analyzer (2006 SEAL Analytical Ltd. Mequon, WI) (Fig. 1).
For tests 1 and 2, Neulog™ dissolved oxygen sensors, Neulog NO3− sensors, and Neulog NH4+ sensors were placed for each container to monitor changes in concentration (Fig. 7). These three sensors were combined and installed in each container, and aluminum foil was used to hold the sensors and plants and block sunlight. Data was monitored every 10 min for 20 h and recorded with NeuLog™ dissolved oxygen sensor, nitrate logger sensors and ammonium logger sensors (Neulog brand dissolved oxygen sensor, EISCO Scientific, Rochester, NY, USA).
Trials 1-3 used the same sensors and parameters as Trials 1-2 to monitor constant dynamic nitrogen changes for 96 h (96 h). The growth medium was 1 L of DI water with the same ingredients as trial 1–1 but used 0.57 g L−1 NH4H2Purchase order4 and 1.8 g L−1 TO KNOW3 instead of 0.22 g L−1 K.H.2Purchase order4 and 0.373 g L−1 TO KNOW3. Additionally, 0.57 g L−1 NH4H2Purchase order4 and 1.8 g L−1 TO KNOW3 were applied every 24 h to provide 5 mg L−1 of NO3− and 5 mg L−1 from NH4+. Data were recorded every 30 min for 4 days (96 h).
In trial 2, a completely randomized design was used with 4 repetitions. There were two SOF at different rates, i.e. CaO2 at 0, 1, 2 and 4 g per jar and MgO2 at 0, 2, 4 and 8 g per jar. The seeds were planted in 7.6 cm pots for two weeks and transferred to the 15.2 cm pots (internal volume: 2622 cm3) after germination. SOF, CaO2 or MgO2 was mixed with the soil (about 2,000 cm3 per pot), the mixed soil was added into the 15.2 cm pots, and then the seedlings were transplanted into the pots, respectively. One seedling was planted per pot. Fertilizer rates were based on the Florida Vegetable Production Handbook28. N, P and K application rates were 112 kg ha−1134 kg ha−1 and 134 kg ha−1 for the whole season. After one week, both groups with CaO2 and MgO2 were each divided into two subgroups. One subgroup was individually flooded in 18.9 L buckets filled with water to simulate flood conditions for two days, while another subgroup was not flooded.
Leaf greenness data representing leaf chlorophyll content determined by SPAD meter (Knoica-Minolta, Osaka, Japan), plant height, ADH activities, and shoot biomass were measured when plant flooding started to wither. To collect the yield data, this test was repeated identically and the yields were measured at 55 DAP.
Root ADH activity
In trial 2, two days after flooding, about 1 g of roots (4–5 cm from the tips) were harvested per treatment and placed in a container filled with liquid nitrogen. Root ADH activity was determined using a modification of the method30 as following. The root tips (1 g) were homogenized in an extraction buffer solution (10 ml) composed of 7.88 g L−1 Tris–HCl (pH 8.0), 0.29 g L−1 EDTA, 0.5 μg L−1 DTT, and 0.08 mg L−1 β-mercaptoethanol. Then, the ADH enzyme extraction solution was centrifuged at 4°C at 15,000 rpm for 10 min. 100 μL of enzymatic solution was added to 900 μL of a reaction solution composed of 7.88 g L−1 Tris–HCl (pH 9.0), 0.29 g L−1 EDTA and 0.66 g L−1 NDA. The mixture was incubated in 1.5 mL microcentrifuge tubes in a 30°C water bath for 3 min. To start the measurable reaction, 100 µL of 100% ethanol was added to the 900 µL reaction mixture. Measurements were taken after 15 s and absorbance at A340 was recorded every 15 s for two minutes. After a reaction time of 1 min in the cuvette, a reading at 340 nm was taken with a spectrophotometer (Eppendorf BioSpectrometer®, Hauppauge, NY. USA) to determine the NADH concentration. Activity was calculated using a value of 6.22 mmol−1 cm−1 as the molar extinction coefficient of NADH at 340 nm31.
All statistical analyzes were performed using R Studio statistical software (RSTUDIO, INC., Boston, MA). A one-way ANOVA was applied to test the significance of SPAD readings, plant height, shoot biomass, yield, and ADH activities. Mean comparisons of significant effects were determined using the Tukey HSD tests. The data collected were all subjected to statistical analysis by ANOVA, and the value of (HSD at 5% was calculated to compare both means. The differences between the means and the correlation coefficients were found to be statistically significant at the level p
Declaration of authorization
All plant experiments, including the collection of snap bean material, ‘Bronco’ used as a cultivar, were carried out strictly in accordance with applicable guidelines, regulations and laws.