We used No.1 (M1) Chinese fir (Cunninghamia lanceolata (Lamb.) Hook.) seedlings that were identified in a preliminary study as exhibiting “passive low-P tolerance” that produced high yields despite low soil P concentration relative to the average genotype (Wu et al. 2011). Seedlings used in this study were half-sibs produced in monoculture form the Chinese fir clonal seed orchard at Wuyi State-owned Forest Farm, Zhangping, in Fujian Province, People’s Republic of China, which was established in 1985. Seedlings were cultivated in a greenhouse at the College of Forestry, Fujian Agriculture and Forestry University, People’s Republic of China, with an average temperature of 20.3°C and relative humidity was 78%. Seedlings were watered 3–4 times weekly. Seedlings had relatively consistent growth rates, complete root system, and showed no signs of disease. The average stem diameter at ground level was 2.30 mm and the average plant height was 17.00 cm.
The experiment was conducted in the Isotope Laboratory of the College of Sciences, Nanjing Agricultural University. Plants were grown in polyethylene containers (4.5cm diameter, 30cm depth) in hydroponic culture to ensure full absorption of exogenous P. Each seedling was wrapped with a sponge and fixed at the mouth of the container in a poly-ethylene foam plate with a 2cm diameter seedling hole cut in the middle of the foam plate. The stem-root transition zone of each seedling was wrapped in sponges and fixed in the seedling hole. During the experiment, a 20-min ventilation controlled by an automatic timer was performed every 4 h to ensure a sufficient oxygen supply to the seedlings.
Seedlings were divided between two P concentration conditions: high-P and low-P, with P concentrations set according to the soil available P in southern Chinese fir plantation forests measured by Sheng and Fan (2005), who showed that optimum available P in southern Chinese fir forests was 0.42 mmol/L (high-P) and the limiting value was 0.03 mmol/L (low-P). We used KH2PO4 as the source of P, and high- and low-P treatments were 0.50 mmol·L− 1 KH2PO4 and 0.03 mmol·L− 1 KH2PO4, respectively. The potassium (K) levels of the nutrient solutions used in the different treatments were adjusted with KCl during the experiment based on the modified Hoagland formula of Wu et al. (2011): 127.5 mg·L− 1 KNO3, 122.5 mg·L− 1MgSO4·7H2O, 294.92 mg·L− 1 Ca(NO3)2·4H2O, trace elements (0.71mg·L− 1 H3BO3, 0.02 mg·L− 1 CuSO4·5H2O, 0.055mg·L− 1 ZnSO4·7H2O, 0.4525 mg·L− 1 MnCl2·4H2O, and 0.015 mg·L− 1 H2MO4·4H2O), and an iron salt solution (1.393 mg·L− 1 FeSO4·7H2O and 1.863 mg·L− 1 Na2EDTA). The pH of the nutrient solution was adjusted to 5.5 with NaOH and diluted HCl.
32P is an ideal radionuclide for use in plant physiology and fertilization studies due to its nuclear features: it is a pure beta emitter with the maximum β− radiation energy Emax=1.7 MeV and a half-life of 14.3d. Based on these properties, we assessed plant P at four times during the experiment (0.5d, 1d, 5d, and 15d). We prepared the 32P radioactive solution using a stock solution of 32P-orthophosphate with a radioactive concentration of 4.05×104 Bq·mL− 1 (PerkinElmer, U.S). Each single seedling pot contained 250 mL nutrient solution and 650 µL of 32P-orthophosphate solution, and there were five replicates for each treatment, for a total of 40 seedlings. We also prepared five polyethylene containers without seedlings for each nutrient solution (i.e., high- and low-P concentrations) and 32P-orthophosphate solution, which were used as blanks to determine the specific activity at the end of the experiment.
Determination Of Indicators
Determination of dry weight
After each P treatment, the nutrient solution was washed from the root surface of seedlings with deionized water until the radioactivity of 32P on the root surface was less than 100 counts per minute (cpm), as determined by a liquid scintillation counter (LSC; Beckman LS6500). Then fresh leaf, stem, and root of Chinese fir seedlings were separated and oven-dried at 105°C for 2 h and then at 75°C to constant mass to determine dry mass (DM). Before oven-drying, small fresh samples of each organ were taken and stored at -80℃ for the determination of radioactivity (below).
Determination of radioactivity
Radioactivity determination of total P and different P fractions in seedlings
For each plant, 0.03g crushed samples of each organ (root, stem, and leaf) were weighed for radioactivity determination of total P. We also separately weighed 0.20g fresh samples of root, stem, and leaf, ground them into homogenates, and rinsed them with 4 mL of 5% trichloroacetic acid (TCA) in centrifuge tubes. Then 1 mL 5% TCA was added and samples were centrifuged at 1180×g for 5 minutes. We transferred the resulting supernatants into 25mL volumetric flasks, added 5mL acetone, shook well, added 5mL ammonium molybdate reagent, mixed, let rest for a few minutes, and then them transferred into a separatory funnel. We then added 10mL of a water-saturated mixture of isobutanol and benzene to each sample and vigorously shook. After resting for a few minutes, the solutions separated into two layers: an acidic inorganic P (Pi) bottom layer and organic P compound upper layer. We collected the two layers in 25mL volumetric flasks containing distilled water for Pi and acetone for organic P. After the main component of the organic P solution (ester P) was removed, we extracted the residue with 3 mL of 95% (w⁄v) ethanol. We continued to extract with a total of 3mL 2:1 mixed solution (ethanol and ether, v⁄v) after resting the solution for 10 minutes and centrifuging at 1089×g for 8 minutes. Each extraction resulted in a lipid P supernatant and the extraction solution was adjusted to 25ml with acetone. The precipitate was hydrolyzed with 5mL of 0.5mol KOH at 36℃ for 18h, cooled, MgCl2 was added to accelerate RNA decomposition, and acidified to pH = 1 with 72% HClO4 before centrifuging at 1180×g for 10 minutes. The resulting supernatant contained 32P-RNA, and the volume was fixed to 25 ml with distilled water. A final precipitate was extracted by adding 5 mL 5% HClO4 and incubating at 90℃ in a water bath for 15 minutes. The solution was separated into two layers after centrifuging at 1180×g for 10 minutes, and a supernatant containing 32P-DNA was extracted and brought to 25 ml with distilled water.
The above extraction steps were repeated four times for complete separation, and final P fractions were determined uniformly. All samples were decolorized and filtered with activated carbon, then 1mL was pipetted, and the radioactivity of the samples was determined by liquid scintillation counter (Beckman LS6500) after adding scintillation liquid (PerkinElmer, Boston,U.S) for 12h. Data obtained from LSC were transformed into units of disintegration per minute (dpm) by dividing cpm by LSC efficiency: dpm = cpm/efficiency (Nurmayulis et al. 2013). Data was recorded as the average of five replications. The reference moment chosen for all activity results was the harvesting time. 32P uptake was calculated using the following decay correction.
Where A is the remaining activity of 32P after decay at time t (from the measurement time to the reference time), A0 is the activity of 32P at t = 0, and λ is the decay constant of 32P. The formula for calculating λ is λ = ln2/T1/2, where T1/2 is the half-life of 32P, which is 14.3d.
Determination of activity specific
Specific activity, expressed in units of Bq·µg− 1, was defined as the ratio of radioactivity to P content in the five blank solutions (see Study Design, above) and calculated as follows:
Where SA is specific activity, Rl is the radioactivity of nutrient solution, and mp is P content in nutrient solution.
We calculated the content of exogenous P of each organ on a dry matter basis by dividing the radioactivity of each organ by the specific activity and dry mass:
Where MTP (expressed in units of µg·g–1) is the content of exogenous P of each organ on a dry matter basis, RTP (expressed in units of Bq) is the radioactivity of total P in each organ, SA (expressed in units of Bq·µg–1) is the specific activity (Eq. 2), and mo (expressed in units of g) is dry mass of each organ. Eq. 3 was also used to calculate the content of each P fraction in each organ was calculated similarly.
We also calculated the distribution ratio of different P fractions in each organ to total plant P; the distribution ratios (rFP, %) was calculated as follows:
Where rFP is distribution ratio of a given P fraction in a given each organ, MFP is the content of that P fraction in the organ on a dry matter basis, and M (expressed in units of µg·g− 1) is the sum of MTP in different organs.
Finally, we calculated the P use efficiency (PUE, g·µg− 1) of each organ, to investigate the rate of dry mass synthesis per unit P in each organ:
Statistical analyses were performed in SPSS version 19.0 (SPSS Inc., Chicago, IL, U.S), and graphs were plotted using Origin 8.5 (Origin Lab Corporation, Northampton, MA, U.S). Paired t-tests were used to compare differences between high and low P treatments at each time point. One-way ANOVA was used to evaluate differences between time points for total P concentrations, distribution ratios, relative proportions of each P fraction, and PUE. Pearson correlation analysis was used to examine the relationships among different P fractions and PUE. All tests were done using a confidence level of P < 0.05. We further investigated significant effects with Duncan’s multiple range test at 5% significance level to test for differences between treatments.