LDH-lactate-NS with different particle sizes, charge properties, and adsorption capacity can be obtained at different titration temperatures of the co-precipitation reaction
TEM images of LDH-lactate-NS, which was synthesized and delaminated by titration reaction at 0°C, 15°C, and 25°C, are shown in Fig. 1A, 1B, and 1C, respectively. As shown in Fig. 1D, the zeta potential analysis of LDH-lactate-NS obtained at 0°C, 15°C, and 25°C showed that the zeta potential was unimodal, indicating that only one charged particle existed in the solution. Given that the zeta potential of LDH-lactate-NS synthesized at 0°C, 15°C, and 25°C was 17.4 mV, 30 mV, and 40.3 mV, respectively, it is reasonable for us to propose that the stability of the solution is LDH_0°C < LDH_15°C < LDH_25°C. As shown in Fig. 1E and Additional file 2: Table S2, the main particle sizes of LDH-lactate-NS synthesized at 0°C, 15°C, and 25°C were 38.2 nm (92%), 42.6 nm (94.3%), and 61.2 nm (96.3%), respectively. Within the titration temperature of the co-precipitation reaction at 0°C, 15°C, and 25°C, the particle size and solution stability of LDH increased with increasing temperature of the titration of the co-precipitation reaction. At low temperatures, LDH-lactate-NS with relatively small particle size and relatively low zeta potential can be obtained.
The DNA adsorption behavior of the LDH-lactate-NS obtained at different temperatures was studied. The DNA adsorption on LDH-lactate-NS was investigated using electrophoresis analysis, as shown in Fig. 1F. The positive control was a DNA solution with the same mass and concentration, and the adsorption gradient was 1:1 to 1:10 according to the mass ratio of DNA:LDH-lactate-NS. After adsorption for 30 min, gel electrophoresis was performed for detection. Fig. 1F shows that the mass ratio of fully absorbed DNA on LDH-lactate-NS synthesized by titration at 0°C is DNA:LDH=1:7, while the mass ratio of fully absorbed DNA at 15°C is DNA:LDH=1:5, and that at 25°C is DNA:LDH=1:4. The results showed that LDH-lactate-NS synthesized at 25°C had the highest DNA adsorption capacity of the three LDH-lactate-NS. The higher the titration temperature, the stronger the adsorption capacity.
To further investigate the effect of contact time on the adsorption of DNA by LDH-lactate-NS obtained at different co-precipitation reaction temperatures, we used various adsorption times of 1, 5, 10, 20, 30, and 60 min in the adsorption experiments. Fig. 1G shows that the adsorption rates of DNA gradually increased with the increase in contact time. After 1 min, the DNA adsorption rate of LDH-lactate-NS obtained at 0°C was only 0.13%, while the DNA adsorption rate of LDH-lactate-NS obtained at 15°C and 25°C was 13.21% and 25.81%, respectively. After 5 min, the DNA adsorption rate of LDH-lactate-NS obtained at 0°C, 15°C, and 25°C reached 0.98%, 16.43%, and 28.57%, respectively. After 60 min, the DNA adsorption rate of LDH-lactate-NS obtained at 0°C, 15°C, and 25°C was 13.56%, 34.11%, and 44.24%, respectively. After 5 min, LDH-lactate-NS synthesized at 15°C and 25°C adsorbed DNA, while in contrast, LDH-lactate-NS obtained at 0°C adsorbed little DNA. In all, the results indicated that with the increase in adsorption time, the DNA adsorption rate of LDH-lactate-NS gradually increased. However, the DNA adsorption rate of LDH-lactate-NS obtained at 25°C was much higher than that of LDH-lactate-NS obtained at 0°C and 15°C.
The ability of LDH obtained at different temperatures to deliver negatively charged fluorescent dye into intact plant cells
In this study, we used BY-2 cells (Fig. 2A) as model systems to investigate the ability of LDH-lactate-NS obtained at different temperatures to act as molecular carriers for plant cells. The neutral nano-platelet conjugate LDH-lactate-NS-FITC was able to shuttle the negatively charged fluorescent dye FITC, which is membrane-impermeable, into the cytosols of the intact plant cells for 10 min. It should be noted that in the previous study, LDH was obtained at room temperature. As shown in Fig. 2B and 2C, after 15 min, there was no fluorescence in the CK and LDH-lactate-NS solutions alone, while in Fig. 2D, a large amount of negatively charged FITC was enriched outside the cell wall of BY-2 cells, showing a diffuse distribution. As shown in Fig. 2E and 2F, the fluorescence of LDH-lactate-NS-0°C+FITC and LDH-lactate-NS-15°C+FITC was weak. As shown in Fig. 2G and 2H, after the mixture of LDH-lactate-NS-25°C+FITC was added to the medium and the cells were treated for 15 min, negatively charged FITC gathered at the nucleus in the cells. The results showed that LDH-lactate-NS-25°C adsorbed FITC, penetrated the BY-2 cell wall, and aggregated in the nucleus within 15 min, with faster and more effective action as compared to the other two materials.
LDH-lactate-NS does not affect the germination rate and promotes root growth
To understand how different concentrations of LDH-lactate-NS obtained at 25°C (LDH-lactate-NS for short) would affect cell division in roots, we measured the germination rate and the root length with and without LDH-lactate-NS or with RM, which consists of the raw materials of LDH-lactate-NS. As presented in Additional file 3, both LDH-lactate-NS and RM affected the A. thaliana seed germination rate. After growing in the culture room at 25°C for 3 days, we found that LDH-lactate-NS did not affect the seed germination rate (all >95%) in the concentration range of 1–300 µg/mL (P<0.05), whereas RM strongly inhibited seed germination at a high concentration. After 3 days, the germination rate for seeds treated with 100 µg/ml RM was 81.58%, and then, 94.64% after 4 days.
Based on the fact that LDH-lactate-NS did not affect the seed germination rate, whereas high concentrations of RM inhibited seed germination, we speculated that LDH-lactate-NS and RM exerted different biological effects on seed germination. Thus, we measured the root length of A. thaliana growing for 5 days (Fig. 3A and 3B). A more modest activation of root length growth (2.97 ± 0.24 cm) was observed for the root exposed to LDH-lactate-NS at low concentrations (1 µg/mL), while a significant increase (5.13 ± 0.64 cm) in cell growth was observed for high doses (100 µg/mL). Then, there was a mild decrease (3.51 ± 0.17 cm) at high concentrations (300 µg/mL), but it was still higher than the growth measured for the wild-type (2.89 ± 0.20 cm). On the contrary, we found that the RM decreased growth with reduction of the concentration of 1–300 µg/mL, as shown in Fig. 3A and 3B, with almost no root elongation (0.15 ± 0.07 cm) at high concentrations (300 µg/mL). Thus, the addition of LDH-lactate-NS to the medium resulted in an increase in root length, but the addition of RM resulted in a decrease in root length.
LDH-lactate-NS affects the expression of genes involved in root cells
On the basis of previous findings, we suggested that LDH-lactate-NS can affect the expression of a number of genes that are essential for cellular functions. To test this hypothesis, we monitored the expression of genes essential for growth in plants (such as aux1, pin1, pin2, and pin3) in Arabidopsis root cells grown on medium supplemented with 1–300 µg/ml LDH-lactate-NS or 1–100 µg/mL RM (Arabidopsis cannot grow in 300 µg/ml RM), as well as on regular MS medium (CK). As shown in Fig. 4A, compared with CK, the expression levels of aux1 and pin1 in RM were lower than that of CK in the range of 1–100 µg/mL, and the relative expression levels decreased with increasing concentration. There was a similar trend in the expression of aux1 and pin1 genes in LDH-lactate-NS. Notably, when 1 g/mL LDH and 10 g/mL LDH were used, the expression level of aux1 relative to CK was 3.07 ± 0.33-fold and 1.47 ± 0.20-fold, and the expression level of pin1 relative to CK was 1.90 ± 0.19-fold and 1.04 ± 0.07-fold, respectively. The results showed that at a low concentration of LDH (1–10 µg/mL), the expression levels of aux1 and pin1 were higher than those of CK in the roots of A. thaliana seedlings that had grown for 5 days. With the increase in the LDH concentration, the expression of the aux1 and pin1 genes decreased.
At 1 µg/mL RM, the expression of pin2 was similar to that of CK, and the relative expression of pin2 increased with increasing RM concentration. At 100 µg/mL RM, the relative gene expression of CK increased to 3.57 ± 0.23-fold. PIN2 gene expression at 1, 10, 100, and 300 µg/mL LDH was 1.39 ± 0.10-fold, 1.23 ± 0.12-fold, 1.26 ± 0.11-fold, and 1.16 ± 0.15-fold, respectively, compared with CK. The results showed that the relative expression of the pin2 gene in the roots of A. thaliana seedlings treated with different concentrations of LDH-lactate-NS was slightly higher than that of CK, and the relative expression of the pin2 gene decreased with increasing LDH-lactate-NS concentration. As shown in Fig. 4A, the relative expression of pin3 was higher than that of CK regardless of the addition of LDH-lactate-NS or RM. In roots with different concentrations of LDH-lactate-NS, the expression of these genes was associated with auxin transport, and therefore, we measured the auxin flow and auxin content. The results showed that the distribution of PIN2pro:PIN2-GFP did not change after different concentrations of LDH-lactate-NS were added, even at fairly high concentrations (1,000 µg/ml) (see Fig. 4B).
With the increase in LDH-lactate-NS concentration, the auxin content and auxin flux increased at 0–0.6 mm
To determine the possible mechanism for the promotion of root growth and altered geotropism responses to 1, 10, 100, and 300 µg/ml CK and LDH, we further measured auxin flux profiles in vivo in the root apical region using a noninvasive microelectrode system, which indicated that all lines have a net rhizosphere auxin flux in the root tip region (Fig. 5A and 5B). The peak auxin flux occurred at 0.2 mm from the root apex in 1, 10, 100, and 300 µg/ml CK and LDH-lactate-NS (Fig. 5A). The flow rates of 1 µg/ml CK (7.54 ± 0.49 fmol·cm−2·s−1) and 1 µg/ml LDH (6.36 ± 3.48 fmol·cm−2·s−1) were similar, at 0.2 mm from the root apex, whereas the flow rates of 10 µg/ml LDH (14.56 ± 3.90 fmol·cm−2·s−1), 100 µg/ml LDH (16.65 ± 3.32 fmol·cm−2·s−1), and 300 µg/ml LDH (18.45 ± 4.21 fmol·cm−2·s−1) were significantly increased (P<0.01). The results showed that at 0.2 mm from the root apex, the flow rate of IAA increased with increasing LDH-lactate-NS. At 0.4 mm from the root apex, 1 µg/mL CK (−3.45 ± 1.51 fmol·cm−2·s−1) and 1 µg/mL LDH (−1.97 ± 0.68 fmol·cm−2·s−1) exhibited similar auxin influx. In contrast, 10 µg/mL LDH (5.29 ± 0.72 fmol·cm−2·s−1), 100 µg/mL LDH (13.80 ± 3.92 fmol·cm−2·s−1), and 300 µg/mL LDH (12.94 ± 2.77 fmol·cm−2·s−1) exhibited auxin efflux. The auxin efflux of 100 µg/mL and 300 µg/mL LDH were similar and higher than that of 10 µg/mL LDH. The auxin flow trend at 0.6 mm from the root tip was similar to that at 0.4 mm, as shown in Fig. 6A.
The trend of auxin flux was consistent with that of CK in the root meristem and transition zone (0–0.6 mm) treated with the low concentration of LDH-lactate-NS (1 µg/mL). At higher concentrations (> 10 µg/mL), auxin flux showed a significant efflux trend, and the peak value increased with increasing LDH-lactate-NS concentration. These results suggest that the rootward localization of LDH-lactate-NS in meristematic cortical cells exerts a negative regulatory effect on auxin transport, and with increasing LDH-lactate-NS concentration, auxin flux decreased.
To quantitatively and qualitatively analyze the effects of LDH-lactate-NS on auxin content in Arabidopsis roots, we used ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to analyze plant hormones. Mass spectrometric analysis (Fig. 5C) showed that the amount of IAA in 1, 10, 100, and 300 µg/mL CK and LDH was 0.26 ± 0.05 ng/g, 0.26 ± 0.03 ng/g, 0.36 ± 0.01 ng/g, 0.48 ± 0.06 ng/g, and 0.39 ± 0.07 ng/g, respectively. The auxin in Arabidopsis roots increased with the addition of LDH-lactate-NS. In the range of 1–300 µg/mL, the root auxin content of Arabidopsis increased with increasing LDH-lactate-NS concentration, with the greatest increase when 100 µg/mL LDH was added to the medium (P<0.01).