Plant growth
Two leaf stage cotton plants (Xinluzao 74, XLZ 74) were grown in Hoagland solution. Seeds were sown in pots (10×10 cm) filled with standard soil mix (Xingyuxing, Wuhan, China). After cotyledons unfolded, uniformed plants were transplanted into a tray filled with Hoagland solution. Plants were grown in growth room at 200 μmol m-2 s-1 photosynthetic active radiation (PAR), 28±1 ˚C and 25±1 ˚C at day- and night-time, respectively. Relative humidity was maintained at 70%, and day/night regime was 14 h/10 h. Hoagland solution was refreshed once every five days. After cotton plants reached the two true leaf stage, six uniform plants were selected and transplanted in a tray with 5 L Hoagland solution before treatment. For the salinity stress, NaCl salt was added into the culture solution of the tray to make the salt level to reach 200 mM.
Synthesis and characterization of PNC
The synthesis and characterization of poly (acrylic acid) coated cerium oxide nanoparticles (PNC) were followed the method described in our previous publications [33, 34]. Briefly, 4.5 g poly (acrylic acid) and 1.08 g cerium(Ⅲ) nitrate were respectively dissolved in 5.0 mL and 2.5 mL deionized water, and the two solutions were mixed thoroughly at 2, 500 rpm for 15 min using a vortex mixer. The mixture was then added dropwise to 15 mL ammonium hydroxide solution (30 %) in a 50 mL beaker and kept stirring at 500 rpm for 24 h at 25 ˚C. Then, the solution was centrifuged at 4, 000 rpm for 1 h to remove any debris and large agglomerates. With the centrifugation at 4, 500 rpm for six cycles, 10 K Amicon cells were used to collect the supernatant which is purified from free polymers and other reagents. The final PNC solution was stored in a refrigerator (4 ˚C) for two weeks. The absorbance of final PNC solution at 271 nm was measured by the UV-VIS spectrophotometer, and the concentration was calculated using Beer-Lambert’s law. All chemicals are from Sigma Aldrich, unless otherwise specified.
The hydrodynamic diameter (DLS size) and zeta potential were determined by 90 Plus PALS (Brookhaven Instruments Corporation, USA). 20 μL of PNC (0.45 mM) was mounted on a holey carbon-coated copper grid, and the PNC TEM imaging was done by a FEI Talos microscope operating at 300 kV.
DiI labeling of PNC
The labelling of PNC with DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine) was followed the method described in our previous publications [33, 34]. Briefly, in a 20 mL glass vial, 4 mL, 0.5 mM PNC and 200 μL, 0.3 mg/mL DiI (in DMSO) was mixed at 1000 rpm for 1min. The resulting mixture was purified using 10 kDa filter (4, 500 rpm for 5 min at least five times) to remove the free chemicals. The final solution was labelled as DiI-PNC and was stored in a refrigerator at 4 ºC for further use.
Foliar delivery of PNC and DiI-PNC to cottonplant
Foliar delivery of PNC and DiI-PNC to cotton leaves was followed the method of our previous publication [35] with minor modifications. Briefly, PNC and DiI-PNC formulation were complexed with the surfactant Silwet L-77 (0.05%, Yuanye, Shanghai, China). 0.1 mL, 0.9 mM PNC and DiI-PNC were foliar delivered to each leaf by using a 1000 μL pipette. The 1000 μL pipette tips was cut about 0.3 cm from the top to remove the sharp tip to avoid the possible physical damage during the foliar spraying. The excess solution on the sprayed leaf were removed immediately. After the spraying, cotton leaves were incubated in the room light for 3h to allow the incubation and plant adaption.
Laser confocal microscopy imaging
To quantify ROS in vivo, leaf discs (diameter, 5 mm) from the first and second true leaves of the stressed plants (200 mM NaCl, 5 days) were incubated with 25 μM 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA, staining of H2O2) or 10 μM dihydroethidium (DHE, staining of •O2—) dyes in 1.5 mL tubes for 30 min under darkness. Confocal imaging was performed as described in our previous publication [33] with modifications. After the above-mentioned 0.5 h incubation, the leaf discs were mounted on microscope slides with mounting medium and sealed with a coverslip. Leaf discs were imaged by using Leica SP8 spectral confocal laser scanning microscope. The confocal microscope was manually focused on a region of leaf mesophyll cells. Three individuals (2 leaf discs for each plant) in total were used. The imaging settings were as follows: 488 nm laser excitation; PMT1, 500-600 nm, for DCF and DHE fluorescence; PMT2, 700-785 nm, for chloroplast fluorescence. ROS imaging with DCF and DHE was analysed with Fiji.
The visualization of DiI-PNC in cotton leaves was also performed by using Leica SP8 spectral confocal laser scanning microscope. Briefly, after 3h incubation of the foliar delivered DiI-PNC with the leaves of the cotton plant, leaf discs (diameter, 5 mm) from the first and second true leaf were made and mounted on the glass slides. PFD (perfluorodecalin) were used to enable better imaging quality. After sealing the slides with a coverslip, the samples are ready for confocal imaging. The imaging settings for DiI-PNC visualization in cotton leaves were as follows: 514 nm laser excitation; PMT1, 550-615 nm, for DiI-PNC fluorescence; PMT2, 700-750 nm, for chloroplast fluorescence. Colocalization between DiI-PNC and chloroplasts was analysed with LAS AF Lite software follow the method described in our previous publications [33]. Three lines of sections were drawn across the ROI (region of interest) with 40 μm interval on the confocal images. The colocalization rate between PNC and chloroplasts was recorded by calculating the proportion of DiI-PNC fluorescence which are overlapped with chloroplast fluorescence emission peaks out of all chloroplast peaks.
Cotton plant performance under salinity stress
The chlorophyll content index (CCI) of the first and second true leaves was daily monitored in salt stressed cotton plant (200 mM NaCl, 5 days). CCI measurements were performed using a chlorophyll meter (SPAD-502 PLUS, Konica Minolta, Japan) with each leaf being measured at three different points (each data point was composed of at least three CCI readout). Plant height was also daily measured for 5 days after the onset of salinity stress. Plant height is defined as the distance between the growth point of the top leaf and the ground [36]. Biomass was determined after 5 days salinity stress. Plants were drying firstly at 105 ºC for 30 min, and then at 85 ºC for 72 h until reaching the constant weight. The phenotype images of the salt stressed cotton plants with and without PNC were taken by a Nikon D810 camera.
Determination of hydrogen peroxide (H2O2), superoxide anion (O2•—) and malondialdehyde (MDA) content
Measurement of H2O2 content in leaf sample was done following a widely accepted method [37] with minor modifications. Approximately 200 mg of fresh samples were placed in liquid nitrogen and then ground with 2 mL of cold acetone. The mixture was then centrifuged (3, 000 rpm) for 15 min. 1 mL of Ti(SO4)2 (5%/W/V in concentrated HCl) was added to the supernatant. After shaking, the samples were then centrifuged (3, 000 rpm) and the precipitates were solubilized in 1 mL H2SO4. The absorbance of the final solutions was measured at 415 nm vis UV-Vis spectrophotometer.
Estimation of superoxide anion radicals (O2-) was done following a widely accepted method [38] with minor modifications. NH2OH was used as a probe for O2-, being oxidized to NO2-. NO2- can react with α-naphthylamine and sulfamic acid to turn the mixture to red colour. Then, the absorbance of the mixture can be measured at 530 nm by UV-Vis spectrophotometer. The method described in previous publication [39] was followed for the assessment of MDA content. Briefly, 200 mg of samples were homogenized with 5 mL TCA having 0.25% 2-thiobarbituric acid (TBA). After incubating at 90 ºC for 30 min, the mixture was immediately cooled down, and then was centrifugate at 8000×g for 15 min. The absorbance of the solution was measured at 450 nm, 532 nm and 600 nm.
Measurement of antioxidant enzymes activities
To determine the activity of SOD, CAT and POD, the first and second leaves of the salt stressed cotton plants (200 mM NaCl, 5 days) were separately collected. For the determination of SOD activity, the freshly collected leaf samples were ground with PBS buffer (pH 7.8). The supernatant was collected following the centrifugation at 12, 000 g for 20 minutes. The supernatant was mixed with EDTA-Riboflavin, NBT and methionine. The mixture was incubated under 200 μmol m-2 s-1 lux light for 20 min. The absorbance of the final mixture was measured at 560 nm by a UV-Vis spectrophotometer (UV 1800PC, AOE, Shanghai, China). SOD enzyme activity was calculated by using the measured absorbance value following the equation 1:
SOD activity=(Ab-As)×VT/(Ab×0.5×Ws×Vs).
Where Ab and As are the blank control and samples’ absorbance value, respectively. VT is the volume of crude leaf extraction mixture. Ws is fresh weight and Vs is the used volume during the sample measurement.
For the determination of POD activity, the collected samples were ground with PBS buffer (pH 5.5). The supernatant was collected following the centrifugation at 3000 rpm for 10 minutes. For POD activity measurement, the solution was prepared with mixing the supernatant of the crude leaf extraction mixture, 20 % TCA, 50mM guaiacol solution, PBS buffer (pH 5.5, 50mM), and 2 % H2O2. The POD activity was measured by calculating the averaged decrease of the recorded absorbance value at 470 nm (1 record/1 min, 3 min) by a UV-Vis spectrophotometer (UV 1800PC, AOE, Shanghai, China). The calculated average decrease value was used to calculate the POD enzyme activity following the equation 2:
POD activity=A470×VT/(W×Vs×t×0.01).
Where A470 is the averaged decrease of the recorded absorbance value at 470 nm. VT is the volume of the crude leaf extraction mixture. W is fresh weight. Vs is the used volume during the sample measurement. t is the reaction time and 0.01 was defined as the unit enzyme activity.
For CAT activity measurement, the collected samples were ground with PBS buffer (pH 7.8). The supernatant was collected following the centrifugation at 4,000 rpm for 15 minutes. The supernatant of the crude leaf extraction mixture was vortexed with PBS buffer (pH 7.8) and 10 mM H2O2. The CAT enzyme activity was calculated based on the averaged decrease of the recorded absorbance value at 240 nm (1 record/1 min, 4 min) by a UV-Vis spectrophotometer (UV 1800PC, AOE, Shanghai, China). Then, the measured absorbance value was used to calculate the CAT enzyme activity following the equation 3:
CAT activity=A240×VT/(W×Vs×t×0.1).
Where A470 is the averaged decrease of the recorded absorbance value at 240 nm. VT is the volume of the crude leaf extraction mixture. W is fresh weight. Vs is the used volume during the sample measurement. t is reaction time and 0.1 was defined as the unit enzyme activity.
Measurement of chlorophyll content and photosynthetic parameters
Leaf samples (the separated first and second leaves) was mixed with a solution containing acetone and ethanol (1:1) for 24 h at dark condition on a shaker (50 rpm). Following centrifugation at 2000 rpm, 10 min, the supernatant was collected. By using a spectrophotometer, the absorbance of the supernatant was measured at 644 nm and 662 nm for the determination of the content of chlorophyll a and chlorophyll b. The chlorophyll a and b content were calculated using the following equations:
Equation 4: Chlorophyll a content = 9.784 × A662 – 0.99 × A644.
Equation 5: Chlorophyll b content = 21.426 × A644 – 4.65 × A662.
Where A662 and A664 are the absorbance value measured at 662 nm and 644 nm, respectively.
Photosynthesis rate, intercellular CO2 concentration, stomatal conductivity and transpiration rate of the first and second leaves were measured by using a portable photosynthetic apparatus Li-6400 XT at D0 (200 mM NaCl, day 0) and D5 (200 mM NaCl, day 5). The measurement settings were set as: 1500 μmol m-2 s-1 photosynthetic photon flux density, 400 μmol mol-1 CO2 concentration, and 25 ºC leaf temperature.
Estimation of K+ and Na+ content
For the estimation of leaf K+ and Na+ content, the first and second leaf samples were milled with a grinder and filtered through a 0.5 mm sieve to collect the grounded samples. 0.2 g of grounded samples were digested for 1.5 h in concentrated H2SO4 (18.4 M). After cooling down, 30% H2O2 was added into the digested samples to get the transparent mixture solution. The mixture was digested for another 1 h to make sure the H2O2 decompose completely. Flame photometer (FP6431, Jiangke, Shanghai, China) was used to determine the content of K+ and Na+ in the samples. The setup of standard curve can be found in the literatures elsewhere.
qPCR
Total RNA was isolated using the RNAprep Pure Plant Kit (DP441, Tiangen, Beijing, China). 2 μg of total RNA was reverse transcribed into cDNA using the TRUEscript first Strand cDNA Synthesis Kit (PC5402, Aidlab, Beijing, China). The amplification of qRT-PCR products was performed in a reaction mixture of 12.5 μL SYBR Green qPCR Mix (PC3302, Aidlab, Beijing, China) according to the manufacturer’s instructions. The qRT-PCR analysis was performed on the Bio-Rad CFX Connect Real-Time PCR System (Bio-Rad, California, USA). Three biological replicates and three technical replicates was used for each investigated gene. The relative gene expression was calculated using the 2−ΔΔCt method. The primers used for qRT-PCR are shown in the Table S1 [40-42].
Statistical analysis
All data were represented as mean ± SE and analysed using SPSS 23.0. Comparisons were performed by either one-way ANOVA based on Duncan’s multiple range test (two tailed) or independent samples t-test (two tailed). * for P < 0.05, ** for P < 0.01, and *** for P < 0.001. Different lowercase letters mean the significance at P < 0.05.