Plant materials and phenotypic analysis during seedling treatments
LY9348 seeds were submerged in 75% ethanol for 1 min and disinfected with 0.15% HgCl2 for 1 min. After washing with sterile water six times, the seeds were germinated in Petri dishes in the dark for three days at 37°C. Next, healthy and uniform seedlings were chosen and soaked in a series of Yoshida solutions (1 L, 6 plants per pot) containing 0, 20 µM selenite, and 20 µM Se-AAF for 2 weeks. The pH of the solution was adjusted to 5.5 by HCl or NaOH. Each treatment was replicated in three pots and the solution was changed every three days. Two weeks later, primary root length, shoot length, number of lateral roots, and fresh weight of the shoots were measured.
Field experiments
A replicated field experiment was conducted in 2018 in two places, Lingshui (18°31'47.1"N 110°03'34.9"E, Hainan) and Ezhou (30.3756°N, 114.7448°E, Hubei). The soil properties of Lingshui and Ezhou were as follows: pH, 5.75 and 8.21; available nitrogen, 93.4 mg/kg and 103.9 mg/kg; total selenium, 0.21 mg/kg and 0.32 mg/kg, respectively. The seeds of rice variety “LY9348” (Oryza sativa L.) were purchased from Guoying Seed Industry Co., Ltd (Wuhan, Hubei, China).
The foliar spray of selenium was applied as selenite, fermented Se, or Se-AAF at 30 g ha−1. The three Se sources were prepared as 75 mg L−1 solutions and applied to the foliage of rice plants at the late tillering (LT), initial heading (IH), and full heading (FH) stages. The control rice plants (CK) were sprayed with only distilled water. Both in Hainan and Ezhou, the experiment was performed with a randomized complete block design and in three replicates(Table 1). The size of the plot in Hainan was 25 m2 (5 m×5 m), and in Ezhou, it was 20 m2 (4 m×5 m).
Paddy transplantation, irrigation, and other rice farming practices were carried out based on the farmers’ experience. The transplanting dates were February 2, 2018, and June 18, 2018, while the harvest dates were May 18, 2018, and September 25, 2018, in Hainan and Ezhou, respectively.
Table 1 The details of Se foliar spray experiments conducted in Hainan and Ezhou during 2018.
|
Sowing date and
transplanting date
|
Foliar spray date
|
Harvesting date
|
Plant number and size of one plot
|
Se applied
|
Se working fluid
|
Hainan
|
Jan. 2nd, 2018
Feb. 2nd, 2018
|
Mar. 5th, 2018
Late
tillering stage
|
Mar. 16th, 2018
Initiate heading stage
|
Apr. 17th, 2018
Full heading stage
|
May 18th, 2018
|
576 plants
5 m X 4 m
|
30 g ha−1
1)Selenite
2)Fermented-Se
3)Se-AAF
|
75 mg L−1
|
Ezhou
|
May 22nd ,2018
Jun. 18th ,2018
|
Jul. 20th, 2018
Late
tillering stage
|
Jul. 28th, 2018
Initiate heading stage
|
Aug.24th, 2018
Full
heading stage
|
Sep. 25th, 2018
|
500 plants
4 m X 4 m
|
Sample preparation
Eight rice plants were selected randomly from each plot and divided into four parts, including root, shoot, spike axis, and grain. After washing with distilled water, each part of the rice plant was oven-dried at 60°C to obtain a constant weight and subsequently grounded into powder for measurement of the selenium content.
Three regions were randomly selected from each plot, and grains from 15 rice plants belonging to each region were sampled. After mixing, the husk was removed to separate the brown rice, whose selenium content was measured. Additionally, the protein from the brown rice was extracted, and its selenium concentration was measured. Later, the grain yield and total biomass from additional 100 rice plants per plot were also measured.
Measurement of Se concentration
The rice samples were digested by adding HNO3-HClO4 (Volume ratio = 9:1), with the temperature being maintained at approximately 180°C. The digested solution was restored with 6 mol L−1 HCl, cooled, and filtered at a set volume.
Organic selenium concentration was determined using the cyclohexane extraction method (Sun et al. 2013). The obtained data indicated inorganic selenium concentration; however, the organic selenium concentration could be obtained indirectly by subtracting the inorganic selenium content from the total selenium content.
To determine the protein Se concentration, 30.00 g of brown rice flour was first weighed and added to a 250 mL Erlenmeyer flask, to which 150 mL of 0.2% NaOH (aq) (brown rice powder and sodium hydroxide solution at a ratio of 1:5) was added. The solution was then stirred with the glass rod evenly and placed on a shaker at 40°C. After shaking for 1 h at 100 rpm, the solution was centrifuged at 3800 rpm for 10 min. Then, the supernatant was collected, and the step was repeated to extract the complete protein. The two supernatants were pooled together, and 0.1 M HCl was added to it. The pH was adjusted to an isoelectric point of 4.8 (Souza et al. 2016). The mixture was placed in a refrigerator for 1 h at 4°C and centrifuged at 3800 rpm for 15 min at 4°C. The obtained white precipitate was the protein which was washed thrice with distilled water and centrifuged at 4000 rpm for 5 min to remove the impurities. Later, the precipitate was put in an oven (AFD-270L-200, AoFeiDa Instrument and Equipment Co.,230 Ltd., China) and baked for 36 h at 30°C.
The Se concentration in the filtrate was measured using hydride generation-atomic fluorescence spectrometry (AFS-230E, Kechuanghaiguang Instruments Co., Beijing, China). The soil pH, alkali nitrogen, and total selenium concentration were commissioned by the Hubei Provincial Geological Experimental Testing Center. The instrument used was AFS-820.
The selenium speciation analysis of brown rice was conducted as follows: The brown rice was first hydrolyzed using protease XIV and cellulase for 18 h in a 37°C water bath. The protein was extracted using a methanol-chloroform-water three-phase extractant while the separation was performed using high-performance liquid chromatography (HPLC, SHIMADZU-LC-20AT) and analyzed using an inductively coupled plasma mass spectrometer (ICP-MS, Fisher series X2). The selenium standards were as follows: SeMet, SeCys2, SeMeCys, Se4+ and Se6+. More details are described in other studies (Bañuelos et al. 2012).
Determination of the characteristics of the grain quality
To determine the amylose content (AC), gelatinization consistency (GC), gelatinization temperature (GT), and alkali spreading value (ASV), the brown rice powdered samples were scanned using the Near-Infrared Grain Analyzer Perten DA7250. The instrument was switched on for preheating. After calibrating with the reference standard plate, the powdered sample was put into the sample cup; the surface was scratched and placed on the sample stage of the spectrometer for scanning. Note that the sample volume was constant each time, and the test was repeated twice with each sample. According to the accuracy requirements, the average value for the samples was calculated if the ratio of the difference between the two tests resulted in an average value of less than 2%. If the requirements were not met, the samples were re-tested, and the average value was calculated.
The Amino Acid (AA) Content Assay Kit (AKAM001M) and DPPH Free Radical Scavenging Capacity Assay Kit (A153–1–1) were purchased from the Bioxbio and Nanjing Jiancheng, respectively.
Calculation methods
The Se recovery efficiency (%) of the whole plant and Se recovery efficiency (%) of the brown rice were based on the plant Se uptake. More details are described in studies of (Deng et al. 2017).
Statistical analysis
The statistical analysis was performed using GenStat 18 (18th Edition, VSN International Ltd., Hemel Hempstead, UK) and SPSS 20.0. The data were presented as mean ±standard error (SE). The mean values were compared using the Least Significant Difference test at the 0.05 level of probability. The Principle component analysis (PCA), partial least squares-discriminate analysis (PLS-DA), and variable importance in projection (VIP) were conducted using the “Statistical Analysis” module on MetaboAnalyst (Xia and Wishart 2011).