3.1. Uptake of pesticides by rice plants
Many studies have reported that rice roots could take up the selected pesticides and translated them to the shoot. The transpiration rates of rice in the control group and the three pesticide treatment groups were 0.0072, 0.0068, 0.0069, and 0.0064 mL/g/h, respectively, with no significant difference (Fig. S1). Rice seedling growing in pesticide treatment groups was normal compared with that in the blank control group. The concentration of the three pesticides in solution and plant tissues was shown in Fig. 1. In the shoots, the concentration of triadimefon increased to 0.53 mg/kg at 6h, and then decreased slowly to 0.44 mg/kg at 48h (Fig. 1A). The concentrations of tebuconazole and epoxiconazole continuously increased to 0.925 mg/kg and 0.389 mg/kg, respectively, at 48h. Triadimefon had the highest accumulation, but the fastest degradation rate in the shoot. In plant roots, the pesticide residue concentration in the roots increased first and then decreased (Fig. 1B). tebuconazole peaked at 2.73 mg/kg at 12h and then dropped to 2.20 mg/kg at 48h. Triadimefon reached a maximum of 1.43 mg/kg at 6h and then began to decrease to 0.825 mg/kg at 48h. Concentrations of epoxiconazole reached a maximum of 4.26 mg/kg at 24h and then began to decrease, reaching 3.49 mg/kg at 48h. The concentrations of the three pesticides decreased with the increase of exposure time, and the pesticide concentrations in the nutrient solution began to increase from 12h. The final concentrations were 0.881 mg/kg, 1.07 mg/kg and 0.970 mg/kg at 48h (tebuconazole, triadimefon and epoxiconazole). However, the pesticide concentration in the solution decreased first and then increased with the increase of time, which may be due to the decrease of pesticide absorption in plant tissues. With the uptake of pesticides, water also volatilizes with transpiration. When the rate of water transpiration is higher than the absorption rate of pesticides, the concentration of pesticides in the solution begins to rise.
Table 2 and Fig. 2 show the root concentration factor (RCF) and translocation factor (TF) of three pesticides in rice plants. The RCF was used to describe the ability of rice roots to take up a compound from solutions (Wu et al., 2013). The average RCFs of tebuconazole, triadimefon, and epoxiconazole were 3.08, 1.27, and 4.37, respectively. Epoxiconazole has the highest average RCF, followed by tebuconazole and triadimefon. The result of RCFs reveals that tebuconazole was most readily accumulated in plant roots, and triadimefon was the weakest. TF was used to describe the ability of rice roots to transfer compounds to rice shoots (Wang et al., 2011). The average TF of tebuconazole, triadimefon and epoxiconazole were 0.18, 0.38, and 0.05, respectively. triadimefon has the highest average TF, followed by tebuconazole and epoxiconazole. all TFs were smaller than one, which reveals that the three pesticides were more easily accumulated in plant roots and less transported in plant shoots. Among the pesticides, triadimefon was the most readily accumulated in plant shoots, and tebuconazole was the weakest. The order of the three pesticides in TF was the opposite of the order in RCF. RCF is positively correlated with logKow (Table 1) of pesticides and negatively correlated with water solubility (Table 1). Epoxiconazole has the lowest water solubility and the strongest hydrophobicity and is most easily absorbed and accumulated by plant roots. This might be the possible the reason why the RCF value of epoxiconazole is the largest in rice plants. TF was used to describe the ability of rice roots to transfer compounds to rice shoots (Wang et al., 2011). The average TF of tebuconazole, triadimefon and epoxiconazole were 0.18, 0.38, and 0.05, respectively. TFs is negatively correlated with logKow of pesticide and positively correlated with water solubility (Wu et al., 2018). Triadimefon has the highest water solubility and the weakest hydrophobicity and is most easily transferred from root to shoot.
Table 2
Root concentration factor (RCF) and transfer factor (TF) in rice.
Exposure Time (h)
|
RCF
|
TF
|
Teba
|
Trib
|
Epoc
|
Teba
|
Trib
|
Epoc
|
3
|
2.69
|
0.89
|
3.51
|
0.06
|
0.36
|
0.02
|
6
|
3.15
|
1.63
|
4.86
|
0.11
|
0.35
|
0.04
|
12
|
3.75
|
1.64
|
5.20
|
0.13
|
0.35
|
0.05
|
24
|
3.29
|
1.41
|
4.69
|
0.15
|
0.35
|
0.05
|
48
|
2.49
|
0.77
|
3.60
|
0.42
|
0.49
|
0.11
|
Average
|
3.08
|
1.27
|
4.37
|
0.18
|
0.38
|
0.05
|
a, tebuconazol; b, triadimefon; c, epoxiconazole |
Many previous studies reported that the uptake and translocation of pesticides wererelated totheir physicochemical properties. As shown in Table 1 epoxiconazole has the lowest water solubility and the strongest hydrophobicity. On the contrary, triadimefon has the highest water solubility and the weakest hydrophobicity. The ability of plant roots to absorb non-ionic compounds is influenced by the chemical properties of the compounds (Collins et al., 2006; Li et al., 2019). This is consistent with theabove experimental results. organic compounds with larger logKow are easy to be absorbed by plant roots, because of their strong hydrophobicity, they are easier to be absorbed by roots and concentrated in lipid membranes. Less hydrophobic organic compounds are less concentrated in roots and can enter plants and transmit to other locations with transpiration streams (Létondor et al., 2015). After entering the root system, compounds would conduct conduction to the upper part of the plant with transpiration, and triadimefon with higher water solubility would be more easily conducted from the root of the plant and accumulated in the shoots of rice (Pullagurala, 2018). It can be inferred that the combination of multiple physical and chemical factors determines the transfer of pesticides from the external medium to the rice root and other locations.
3.2. Distribution of pesticides in symplast and apoplast
Figure 3 shows that the three pesticides distributed in the symplast and apoplast of root and shoot of rice. The proportions of triadimefon, tebuconazole and epoxiconazole in the symplast and apoplast of rice plants were 15% − 33%, 6% − 31%, 7% − 37% and 67% − 85%, 69% − 94%, 63% − 93%, respectively. The distribution of the three pesticides in the symplast was significantly larger than that in the apoplast. In general, triadimefon, tebuconazole, and epoxiconazole were mainly accumulated in the symplast after entering the plant, indicating that all the three pesticides had strong transmembrane transfer capacity. The proportion of tebuconazole and triadimefon pesticides accumulated gradually in shoot symplast. The accumulation of tebuconazole in symplast of shoot increased at 12 and 24 h. The accumulation of triadimefon in shoots, epoxiconazole in roots and shoots showed little change in apoplast and symplast. In the root, the mean values of the three pesticides accumulated in the symplast and apoplast were 74%, 74%, 66%, and 26%, 26%, and 36% respectively. In the symplast, the proportion of triadimefon and tebuconazole was same, while the proportion of epoxiconazole was largest, and the order was opposite in the apoplast. The results showed that triadimefon and tebuconazole were more readily accumulated in the symplast and epoxiconazole was more accumulated in the apoplast. In the shoot, the mean values of the three pesticides accumulated in the symplast and apoplast were 74%, 91%, 93%, and 26%, 9%, 7%, respectively. The proportion in the symplast followed the order of epoxiconazole > tebuconazole > triadimefon, and the order was opposite in the apoplast. The results showed that epoxiconazole in the shoot was more readily accumulated in the symplast and triadimefon and tebuconazole were more readily accumulated in the apoplast. The accumulation of tebuconazole and epoxiconazole in shoot and apoplast was significantly larger than that in root apoplast, while the accumulation of triadimefon was not significantly different.
Almost all nonionic organic pollutants enter plants by passive absorption, and their migration is driven by transpiration pull (Kvesitadze et al., 2006). Very few organic pollutants with hormonal properties (Phenoxy acid herbicide) can be actively absorbed by plants (Saunders et al., 1965; Devlin et al., 2006). Triadimefon, tebuconazole, and epoxiconazole can be transferred through the apoplast pathway from rice root to other positions by transpiration pull. A large amount of accumulation was found in the rice symplast, indicating that these three pesticides may be actively transported into the cell through the channel proteins on the cell membrane, or only accumulated in the lipid membrane on the cell surface.Besides, the relative proportion of apoplastic and symplastic movement of chemical compounds was also related to the physicochemical properties of pesticides and physiological characteristics of plant tissues (Zhan et al., 2018). It has been demonstrated that organics with small logKow (large solubility) are easy to be transported through the symplast pathway of plants, while organics with large logKow (small solubility) are easy to be transported through the symplast pathway (Li et al., 2020). Epoxiconazole is the most hydrophobic of the three pesticides, its logKow is 3.44 and water solubility is only 6.63mg/L. Tebuconazole and epoxiconazole had strong hydrophobicity and logKow were 2.77 and 2.77. Our results showed that the larger the pesticide logKow was, the easier to accumulate in the symplast in the shoot, while the smaller the logKow was, the more likely it was to accumulate in the apoplast in the shoot. But this order is not significant in the root.
3.3. Subcellular distribution of pesticides in rice tissues
Figure 4 shows the proportions of triadimefon, tebuconazole, and epoxiconazole in the subcellular distribution in roots and shoots. The proportions of triadimefon, tebuconazole and epoxiconazole in the soluble components, organelle and cell wall of rice plants were 24% − 29%, 14% − 26%, 11% − 21%; 22% − 29%, 15% − 26%, 12% − 39% and 41% − 54%, 52% − 72%, and 45% − 77%, respectively. The distribution of the three pesticides in the cell wall was significantly larger than that in the soluble components and organelle. Triadimefon, tebuconazole, and epoxiconazole were mainly accumulated in the cell walls after entering the plant. The proportion of tebuconazole and epoxiconazole pesticides accumulated gradually in shoot cell wall. The proportion of triadimefon in shoot cell wall and the three pesticides in root cell wall showed little change. The proportion of tebuconazole and epoxiconazole pesticides accumulated decreased gradually in shoot soluble components. The proportion of triadimefon in shoot soluble components and the three pesticides in root soluble components showed little change. The proportion of the three pesticides in root and shoot organelles showed little change. In the root, the mean values of the three pesticides accumulated in the soluble components, organelle, and cell wall were 28%, 20%, 14%; 28%, 18%, 22% and 44%, 62%, 63%, respectively. In the shoot, the mean values of the three pesticides accumulated in the soluble components, organelle, and cell wall were 26%, 16%, 13%; 24%, 20%, 18%, and 50%, 64%, 69%, respectively. It is showed that tebuconazole is more easily accumulated in the soluble component and epoxiconazole was more accumulated in the cell wall.
The results were related to the water solubility of the three pesticides. The water solubility of the three pesticides is triadimefon > tebuconazole > epoxiconazole. Wang et al. (2019) found that because of its strong water solubility, the insecticide thiamethoxam was more easily dissolved in the cell fluid, and had a higher proportion in the soluble components of the root and bud cells of young cabbage. The proportion of triadimefon in root cell walls was significantly lower than that of tebuconazole and epoxiconazole. Plant cell walls are mainly composed of polysaccharides, structural proteins, lignin, lectins, mineral elements, and lipids. In solid solution systems, organic chemicals with low solubility are more likely to be separated into solid phases with high lipid content (Collins et al., 2006; Li et al., 2005). Triadimefon was more soluble than tebuconazole and epoxiconazole and was not easily retained during root uptake and transfer. Kang et al. (2010) found that plant organelles contain more lipid components (15 ~ 30%) than cell walls, which are mainly composed of polysaccharides (90%). In general, more hydrophobic compounds are accumulated in plant tissues with higher lipid content (Gao and Zhu, 2004). Compared with the proportion of pesticides accumulated in the cell wall, the three pesticides were mainly accumulated in the cell wall. The reason for this may be associated withthe ability to cross the membrane into the cell. Tebuconazole and epoxiconazole were mainly distributed on the cell wall in rice shoots and leaves, and they were accumulated in trace amounts in organelles and soluble components. This mightbecause after the pesticide is absorbed by the root, tebuconazole, and epoxiconazole with strong hydrophobicity are largely accumulated in the solid phase of the root, and only a small amount of pesticides is transmitted to the shoots through the xylem. Moreover, due to hydrophobicity, they are mainly concentrated in the cell wall, and only a few of them are transported into the cell membrane. Studies have found that dinotefuran, thiamethoxam, and imidacloprid are mainly accumulated in the soluble components of cells, which are relatively easy to transfer from roots to stems and then to leaves (Dettenmaier, et al., 2008). The proportion of difenoconazole in solid subcellular components was higher than that in soluble components, resulting in its accumulation in roots and less translocation in stems and leaves (Ge, et al., 2017). In lettuce with a low concentration of PAE (phthalic acid ester) and a low concentration of PFOA (Perfluoro octanoic acid), root cell walls and organelles were the dominant storage compartmentsfor PAEs and PFOA, resulting in less translocation to buds. Ju et al. (2020) found that the transportation of pesticide from root to stem and stem to leaf was adversely affected by subcellular fraction concentration factor (SFCF) of cell walls and organelles.