Characterization of PNC
A clear peak at 271 nm was observed in the absorbance curve of PNC (Figure S1). The images from transmission electron microscopy (TEM) shows an average PNC core size of 4.7 ± 0.9 nm (Figure 1a). Analysis from a dynamic light scattering instrument (Malvern Zetasizer, Nano) showed that the average size of PNC by intensity was 8.5 ± 0.2 nm (Figure 1b), and the average zeta potential was -43.3 ± 6.3 mV (Figure 1c).
Influence of PNC priming on rapeseed germination and phenotype
The germination rate of rapeseed was not significantly affected by PNC+TES priming under normal growing conditions as compared to TES (or control) priming (Figure 1d). However, a significant difference of the germination rate was observed between PNC+TES priming and the control/TES priming under 200 mM salinity stress from Day2 to Day7 (trail terminated), showing the final germination rate for PNC priming (84%) and TES priming (76%) (P < 0.05). Compared with the TES priming, PNC+TES priming markedly increased fresh weight (54.4 ± 0.3 vs 34.4 ± 0.7 mg/seedling, 41% increase) of the rapeseed seedlings under salt stress (Figure 1e).
Localization of PNC in seeds during priming hours
Seeds primed with DiI-PNC in 10 µM TES buffer for one, three, and eight hours was sampled for visualizing the distribution of PNC in seed coat, cotyledon and radical. The sampling hours (1h, 3h, and 8h) were based on a preliminary experiment in which we measured the water absorbance by seeds. A clear increase of water content was observed in rapeseed seeds during priming hours, showing a sharp increase from 1h to 3 h, and a steady increase from 4h to 8h (Figure 1f). The results indicated that the seed begins to absorb water from the first hour of priming, whereas the peak difference in water absorbance was recorded at 3h and 8h. PNC priming enabled better water absorbance in seeds than the control at 1, 3 and 8 hours (Figure 1g). During the first hour of priming, Dil-PNC signal was only detected in the seed coat (Figure 2a), compared with no Dil-PNC signals were detected in cotyledon (Figure 2b) and radical (Figure 2c). At 3h priming, Dil-PNC signal was observed in both the seed coat (Figure 2a) and cotyledon (Figure 2b), while no Dil-PNC signal was detected in radical (Figure 2c). Further, during 8h priming, Dil-PNC was found in all tissues of the seed, i.e., seed coat, cotyledon, and radical, showing the signal intensity as seed coat > cotyledon > radical (Figure 2a, b, and c). No signals were detected in the control group at 1h, 3h, and 8h priming (Supplementary Figure S3-5).
PNC nanopriming regulated relative expression of AMY genes to enhance α-amylase activity and total soluble sugar content in rapeseeds
Significant differences in α-amylase activity were recorded during the priming hours of PNC priming (P < 0.05). Seed priming with PNC+TES increased α-amylase activity by 175%, 309%, and 295% at 1h (0.130 ± 0.038 vs 0.047 ± 0.003 mg g—1 min—1), 3h (0.341 ± 0.005 vs 0.0883 ± 0.001 mg g—1 min—1), and 8h (0.357 ± 0.048 vs 0.090 ± 0.002 mg g—1 min—1) priming, respectively, compared to the TES priming control (Figure 3a). Consistently, at 1h and 8h, the relative expression level for AMY1 gene was significantly higher in PNC+TES primed seeds than the TES priming control, while a downregulation of AMY1 gene was found at 3h (Figure 3b). The upregulation of AMY2 gene was found at 8h priming with PNC+TES than the TES control, compared with no difference at 1 and 3h priming (Figure 3c). The highest increase in the relative expression values for AMY1 and AMY2 was recorded at 8h priming, showing 169% and 68% increase respectively (Figure 3b and 3c). Compared to TES priming, PNC+TES priming significantly increased total soluble sugar content (TSS) by 177%, 64%, and 58% during 1h (8.8 ± 0.1 vs 3.2 ± 0.2 mg g—1 FW), 3h (10.4 ± 0.2 vs 6.4 ± 0.2 mg g—1 FW), and 8h (13.1 ± 0.2 vs 8.3 ± 0.2 mg g—1 FW), respectively (Figure 3d). This is in accordance with the increased activities of α-amylase in PNC+TES primed seeds than the TES control.
PNC nanopriming modulates ROS level in the seeds and seedlings of rapeseeds
During the first-hour, compared to TES priming, PNC priming showed significantly increased MDA contents (2555 ± 206 vs 2228 ± 39 mg g—1 FW), H2O2 (18.2 ± 1.3 vs 14.2 ± 0.2 μmol g—1 FW), and •O2— (22.3 ± 0.3 vs 19.1 ± 0.3 μmol g—1 FW) by 15%, 28%, and 15%, respectively (Figure 4a-c). Whereas, at 3h and 8 h, PNC priming reduced the ROS contents. At 3h, PNC priming significantly reduced MDA contents (1428 ± 156 vs 1849 ± 132 mg g—1 FW), H2O2 (7.3 ± 0.1 vs 10.5 ± 0.5 μmol g—1 FW), and •O2— (9.3 ± 0.4 vs 18.1 ± 0.3 μmol g—1 FW) by 23%, 31%, and 48% than the TES priming, respectively (Figure 4a-c). Similarly, at 8h, the MDA contents (1144 ± 14 vs 1625 ± 156 mg g—1 FW, 30% decrease), H2O2 (3.0 ± 0.1 vs 10 ± 0.3 μmol g—1 FW, 70% decrease), and •O2— (5.2 ± 0.2 vs 14.1 ± 0.3 μmol g—1 FW, 64% decrease) was significantly lower in PNC+TES primed seeds than the TES control (Figure 4a-c). A significant increase of SOD and POD activities was found in seeds primed with PNC+TES than the TES control (Figure 4d and 4e). PNC priming increased SOD activities in seeds than the control by 52%, 127%, and 53% at 1h (92.6 ± 1.3 vs 60.8 ± 1.3 U g—1 FW), 3h (156.2 ± 1.3 vs 68.7 ± 1.3 U g—1 FW), and 8h (172.1 ± 1.3 vs 112.5 ± 1.3 U g—1 FW), respectively (Figure 4d). PNC priming increased POD activities in seeds than the control by 76% and 53% and 60% at 1h (6.4 ± 1.0 vs 3.6 ± 0.1 μmol tetra-gualacol /mg protein /min), 3h (5.9 ± 0.4 vs 3.8 ± 0.1 μmol tetra-gualacol /mg protein /min) and 8h (7.1 ± 0.3 vs 4.5 ± 0.1 μmol tetra-gualacol /mg protein/min), respectively (Figure 4e). In contrast to the changes of SOD and POD activities, PNC priming resulted significantly lower CAT activity in seeds than the control, showing 43% and 55% decrease at 3h (0.85± 0.07 vs 1.49 ± 0.09 mmol H2O2/mg protein/min), and 8h (0.84 ± 0.07 vs 1.88 ± 0.15 mmol H2O2/mg protein/min), respectively (Figure 4f).
Compared with the non-stress condition, MDA, H2O2, and •O2— contents were significantly increased in salt stressed (200 mM NaCl, 7 days) seedlings of rapeseeds with or without PNC priming (Figure 5a-e). Under non-saline condition, no difference of MDA, H2O2, and •O2— content was found in either shoot or root seedlings of rapeseeds (7 days old) with or without PNC priming. However, under salt stress, PNC primed seedlings significantly reduced MDA (5234 ± 165 vs 6918 ± 100 mg g—1 FW, 24% decrease), H2O2 (23.4 ± 0.6 vs 69.8 ± 1.8 μmol g—1 FW, 66% decrease), and •O2— (3.4 ± 0.3 vs 6.7 ± 0.3 μmol g—1 FW, 50% decrease) content in shoot than the control (Figure a, 5c and 5e). Similarly, compared with the control, 27%, 39%, and 37% decrease of MDA (3395 ± 47 vs 4656 ± 122 mg g—1 FW), H2O2 (13.2 ± 0.7 vs 21.6 ± 1.6 μmol g—1 FW), and •O2— (6.1 ± 0.7 vs 9.7 ± 1.8 μmol g—1 FW) content was found in the root of PNC primed seedlings (Figure 5b, 5d and 5f). Similar to the results of the seeds during priming hours, compared with a decrease in CAT activities (0.94 ± 0.05 vs 1.78 ± 0.03 μmol H2O2/mg protein/min for shoot, and 1.10 ± 0.02 vs 1.60 ± 0.02 μmol H2O2/mg protein/min for root), a significant increase of SOD (183.9 ± 6.6 vs 151.3 ± 8.2 U g—1 FW for shoot, and 131.7 ± 1.2 vs 107.6 ± 5.7 U g—1 FW for root) and POD (6.5 ± 0.6 vs 5.53 ± 0.05 μmol tetra-gualacol /mg protein /min for shoot, and 8.8 ± 0.3 vs 5.6 ± 0.2 μmol tetra-gualacol /mg protein /min for root) activities was found in shoot and root of seedlings primed with PNC+TES than the TES control under salt stress (Figure 6a-e). Under non-saline condition, no significant difference was found between seedlings primed with and without PNC priming (Figure 6a-e). It shows that PNC priming enhanced shoot and root SOD (21% and 22%, respectively) and POD (17% and 49%, respectively) activities in contrast to the control under salt stress conditions (Figure 6a-d), while a decrease of CAT activity by 82% and 31% was observed in the shoot and root compared to the control under salt stress (Figure 6e-f).
PNC nanopriming affected Na+/K+ ratio in rapeseed under salt stress
Compared to non-saline conditions, an increase of Na+ content was found in salt stressed (200 mM NaCl, 7 days) seedlings of rapeseeds with or without PNC priming (Figure 7a-b). However, seedlings with PNC priming showed increased shoot Na+ content by 13% (17.5 ± 0.4 vs 15.6 ± 0.7 mg g—1 DW, Figure 7a) and decreased root Na+ content by 52% (7.0 ± 0.5 vs 14.5 ± 0.6 mg g—1 DW, Figure 7b) than the control under salt stress. An overall decrease of Na+ content was observed in seedlings primed with PNC than the control under salt stress (24.5 ± 0.6 vs 30.8 ± 0.6 mg g—1 DW, Figure S6a). In comparison with non-saline conditions, K+ content in shoot and root of seedlings with or without PNC priming was significantly reduced under salt stress (200 mM NaCl, 7 days) (Figure 7c and 7d). While seedlings primed with PNC maintained higher K+ content in the shoot and root by 31% (3.80 ± 0.06 vs 2.91 ± 0.27 mg g—1 DW, Figure 7c) and 29% (2.9 ± 0.2 vs 2.3 ± 0.1 mg g—1 DW, Figure 7d), respectively than the control (seedlings primed with TES) under salt stress. An overall better maintained K+ content was observed in seedlings primed with PNC than the control under salt stress (6.7 ± 0.2 vs 5.1 ± 0.2 mg g—1 DW, Figure S6b). Not surprisingly, compared to the TES control, seedlings primed with PNC showed significantly reduced Na+/K+ ratio by 10% and 62% in the shoot (4.6 ± 0.2 vs 5.1 ± 0.2, Figure 7e) and root (2.4 ± 0.2 vs 6.3 ± 0.2, Figure 7f) under salinity stress, respectively. An overall decreased Na+/K+ ratio was observed in seedlings primed with PNC than the control under salt stress (3.65 ± 0.07 vs 5.81 ± 0.08, Figure S6c). No significant difference of Na+/K+ ratio in either shoot or root was found in seedlings primed with and without PNC under non-saline condition (Figure 7e and 7f).