Physicochemical properties of NC and NCFe
The morphologies of NC and NCFe particles were visualized by TEM. The morphology of the NC particles was that of rod-like whiskers (Fig. 1a), which was in agreement with previous reports on NCs [26, 30]. When NCs were mixed with FeSO4, the NCFe particles maintained a small, whisker-like morphology with small dots (Fe) on the surface of the NC particles (Fig. 1b). The Z-average hydrodynamic diameter and zeta potential of the NC whiskers measured by DLS were 84.3 ± 0.2 nm and − 47.3 ± 1.7 mV (Fig. 2b), respectively, with a polydispersity index (PDI) of 0.20 ± 0.01 (Fig. 2a). The particle size and zeta potential of NC-Fe were 107.4 ± 3.0 nm and − 9.7 ± 0.4 mV, respectively, with a PDI of 0.36 ± 0.05 (Fig. 2c and 2d).
NC synthesis includes partial esterification using sulfuric acid under appropriate conditions [26]. Hence, sulfonic groups on the surface of NCs can be determined by means of conductometric titration, which was developed by several groups in 1925 [31, 32]. Figure 3 shows a typical V-shaped titration curve of the conductivity of the NCs. The net charge density calculated by the consumption of titrant for sulfate group neutralization was 112 ± 6.4 mmol/kg in this study.
The surface chemistry of charged nanoparticles significantly affects their biological performance. The net charge density of NCs in aqueous suspension is an important parameter to measure biological activity. A well-dispersed suspension can be easily applied on the plant surface by the spraying method. The size and size distribution of a charged nanomaterial in aqueous solution greatly affect the efficiency of spraying. A negative zeta potential value indicates that the nanoparticle was negatively charged. The net charge density of the NCs indicated the magnitude of ionizable groups on the surface of the particles. A higher net charge density or zeta potential of nanoparticles supplies a stronger repel force to suspend particles in solution, providing a great opportunity to chelate Fe ions for the transportation and dispersion of Fe into a biological system or pectin in the plant cell wall [13].
Phenotype of pear leaves treated with NCFe
Figure 4 shows the phenotypic comparison of pear seedlings grown under the Fe deficiency treatments or normal hydroponic culture. Under low - Fe concentrations, the leaves showed visible symptoms of chlorosis (Fig. 4a). When using Hoagland’s nutrient solution containing 1×10− 4 mol/L Fe-EDTA, the seedlings grew well without obvious chlorosis (Fig. 4b). However, as shown in Fig. 4c, after 72 h of treatment with NCFe (T2-T4) and Fe-EDTA (T5), more evenly distributed green color appeared in the leaves, but ferrous sulfate (T1)-treated leaves were only partially recovered with uneven distribution of a green color, and the yellow leaves still showed green spots. Thus, increased Fe uptake was observed in the NCFe treatments.
The rod-like particles of NCs with a negative zeta potential indicated that the particles were negatively charged (Fig. 2b). It has been proven that NCs have a higher adsorption capacity and better binding affinity than other similar materials at the macroscale [22]. When NCs are mixed with Fe2+, the high content of available sulfonic groups on the NC surfaces is utilized as an anchor point for the simultaneous reduction and stabilization of NC-supported Fe2+. The use of NCs as carriers to restore the availability of Fe could be a valuable and sustainable strategy to reduce the impact of Fe chlorosis on pear and/or crops [33]. This phenomenon may be attributed to the increased interaction between the positively charged metal salt and negatively charged NCs due to the presence of sulfonic groups on the NC surface and iron chelating properties [34, 35].
Fe ions bound to anionic NC whiskers could increase Fe transportation capability and promote active Fe migration to Fe-deficient plants. The phenotype of pear leaves treated with NCFe indicated that in the leaves sprayed with ferrous sulfate alone (Fig. 4c, T1), ferrous ions may not be efficiently absorbed or evenly transported to other places. In the leaves treated with NCFe (Fig. 4c, T2-T4), depending on the formulation of NCs to Fe, ferrous ions could be easily transported into plant cells and increase the active Fe and chlorophyll contents in the cell to improve the photosynthetic rate. We therefore assumed that NCs performed as carriers to bring ferrous ions into leaves and distribute evenly; hence, the leaves returned to green more sufficiently. Comparably, the greenness of the Fe-EDTA-treated plants (Fig. 4c, T5) was between those of the ferrous sulfate- and NCFe-treated plants, indicating that the level of re-greening might be dependent on foliar fertilizer formulation and/or facilitators.
However, a relatively high Fe concentration was not necessarily correlated with re-greening rates because the effectiveness and mobilization of Fe may be restricted within the leaf [36]. We observed that NCFe prepared at a charge ratio of 1:3000 was an optimal formulation to control chlorosis. This finding also agreed with a previous report performed by Fernández et al [37]. The release dynamics and metabolic mechanism of NCFe within Fe-deficient plants at the molecular level should be further explored in the future.
Enhancement of the photosynthetic rate of pear leaves
To study the impacts of NCFe on photosynthesis, we mainly explored the effects of NCFe with an optimal NC-to-Fe ratio (T3) and compared them to those of ferric sulfate (T1) on photosynthetic parameters, including the Pn of leaves, Gs, Ci, Tr, VPD, and WUE. As shown in Table 3, the Pn of T1 and T3 was significantly increased by the NCFe treatment compared with that of CK, and the Pn of T3 was 121.7% higher than that of CK and 40.4% higher than that of T1. In addition, the Gs, Tr and WUE of T3 were significantly increased compared with those of the CK, and there were no significant differences in these parameters between the T1 and CK groups. The Ci and VPD of the CK group were higher than those of the T1 and T3 groups, and there was a significant difference in these parameters between T3 and CK but not between T3 and T1.
Photosynthesis is a complex comprehensive photochemical and biochemical process that is not only light-dependent and takes place in the thylakoidal membrane of chloroplasts but also occurs in the stroma of chloroplast fixation, reduction of CO2 and formation of carbohydrates [38]. It has been reported that metallic nanoparticles and oxides can be used as photocatalysts to convert light to energy and promote the photosynthetic rate when associated with the structure and function of plant photosynthesis [39]. However, preventing and controlling iron chlorosis is difficult and often gives poor results, but copper-iron chelation, for example, could enhance the photosynthetic rate in IDC grape leaves [40]. Our results also demonstrated that spraying NCFe could significantly improve the photosynthetic capacity and photosynthetic rate of leaves compared with the treatment of spraying FeSO4.
Table 3
Effects of treatments on the photosynthetic parameters of pear leaves
Treatment* | Pn* | Gs | Ci | Tr | VPD | WUE |
CK | 6.9 ± 0.8 c | 236 ± 23.7 b | 341.3 ± 8.0 b | 4.9 ± 0.3 b | 2.1 ± 0.1 b | 1.4 ± 0.2 b |
T1 | 10.9 ± 0.4 b | 328.3 ± 13.2 ab | 330.7 ± 2.6 ab | 5.9 ± 0.1 a | 1.9 ± 0.1 a | 1.8 ± 0.1 b |
T3 | 15.3 ± 1.8 a | 428.0 ± 49.1 a | 319.0 ± 6.7 a | 5.8 ± 0.1 a | 1.5 ± 0.1 a | 2.7 ± 0.3 a |
*T1, 2 mmol/L FeSO4; T3: chelate prepared at a NC-to-Fe charge ratio of 1:3000. Pn, net photosynthetic rate of leaves; Gs, stomatal conductance; Ci, intercellular CO2 concentration; Tr, transpiration rate; VPD, saturated vapor pressure difference; and WUE, water use efficiency. |
Enrichment of physiological indicators facilitated by NCFe
Chlorophyll content, active Fe content, and chlorophyll fluorescence parameters are major physiological indicators for studying plant physiological processes [41]. In this study, the SPAD value was used to evaluate the effects of NCFe on the chlorophyll content in leaves (Fig. 5). The results showed that after 72 h of treatment, the chlorophyll content increased from high to low in the following order: T3 > T4 > T2 > T5 > T1 > CK. The chlorophyll content enhancement in treatments T2 to T5 was significantly higher than that in T1 and the control. However, there was no significant difference (p < 0.05) among the NCFe treatments from T2 to T4.
Table 4
Chlorophyll and carotenoid contents of leaves after 72 h of treatment
Treatment* | SPAD | Chlorophyll a | Chlorophyll b | Total Chlorophyll | Carotenoid |
CK | 21.6 ± 1.2 c | 0.40 ± 0.003 c | 0.12 ± 0.003 c | 0.52 ± 0.005 c | 0.19 ± 0.007 b |
T1 | 30.1 ± 1.3 b | 0.61 ± 0.034 b | 0.16 ± 0.013 b | 0.78 ± 0.025 b | 0.22 ± 0.019 b |
T3 | 37.3 ± 1.0 a | 0.85 ± 0.096 a | 0.26 ± 0.029 a | 1.11 ± 0.130 a | 0.32 ± 0.033 a |
*T1, 2 mmol/L FeSO4; T3: chelate prepared with a NC-to-Fe charge ratio of 1:3000. All suspensions were diluted with DI water. Means followed by different letters present significant differences at p < 0.05. |
Table 4 showed that the contents of chlorophyll a, chlorophyll b and total chlorophyll of T1 and T3 were significantly increased compared with those of CK, and the contents of chlorophyll a, chlorophyll b and total chlorophyll of T3 treatment were significantly higher than those of T1, which were increased by 112.5%, 116.7%, and 113.5% compared with the CK and 39.3%, 62.5%, and 42.3% compared with T1, respectively. The carotenoid content in the leaves of T3 was significantly increased compared with that of T1 and CK, but there was no significant difference in the carotenoid content between T1 and CK. The results showed that the chlorophyll and carotenoid contents of plants were increased significantly by spraying NCFe compared with those of plants sprayed FeSO4.
The effects of NCFe on the active Fe content in seedling leaves are shown in Fig. 6. After 72 h of treatment, the active iron contents in the T1 to T5 treatments were all significantly increased compared with that of the control. More importantly, the active iron content in the NCFe treatments (T2, T3, and T4) was much higher than that of the FeSO4 (T1) and Fe-EDTA (T5) treatments. In particular, when the chelate prepared with a NC-to-Fe charge ratio of 1:3000 (T3) was applied, the active Fe content of the treated leaves was significantly enhanced compared with all other treatments. More specifically, the active Fe content increased approximately 9 times compared with that of the control. The results demonstrated that NCs had a strong ability to promote the active Fe content in the leaves, so the IDC of pear leaves was obviously controlled and eventually reversed.
Chlorophyll and carotenoids are major light-harvesting pigments. The value of chlorophyll fluorescence describes plant photosynthetic growth and mechanisms. The light energy absorbed by chlorophyll is mainly consumed by photosynthesis, chlorophyll fluorescence and heat dissipation [42]. This study showed that NCFe could increase the chlorophyll contents up to more than 70% compared with the control (Table 4) and strongly promoted chlorophyll fluorescence parameters in IDC leaves (Table S1). A previous study showed that sulfate group density played an important role in the chelation of Fe to NCs and/or in their biological activity [43]. Our results indicated that different concentrations of NCs in the NCFe formulation had different impacts on chlorosis recovery (Fig. 4c). At an appropriate chelation formulation, CNFs could enhance physiological indicators (Fig. 5) and even increase the chlorophyll contents up to more than 70% compared with the control (Table 4). A small increase in chlorophyll and carotenoids comes together to form a reaction center where the absorbed light energy is initially converted into chemical energy, resulting in plant healthy growth [12].
Regulation of relative gene expression in pear leaves
Ferritin plays an important role in iron storage in plants [11]. We mainly studied the relative expression levels of P. betulifolia ferritin genes (PbFER1, PbFER2, PbFER3 and PbFER4) in leaves as well as PME gene expression. The results showed that expression levels of ferritin genes PbFER1, PbFER2 and PbFER3 in the T1 and T3 plants were significantly upregulated compared to those in CK plants after 72 h of treatment, and the relative expression levels of the three genes in the T3 plants were significantly higher than those in the T1 plants (Fig. 7). The expression levels of PbFER1, PbFER2 and PbFER3 in T3 plants were 3.6, 4.2 and 4.0 times those in CK plants and 1.5, 1.6 and 1.7 times those in T1 plants, respectively. The relative expression level of PbFER4 in T3 was significantly higher than that in T1 and CK, but there was no significant difference between T1 and CK. The relative expression levels of PbFER1, PbFER2, PbFER3 and PbFER4 in leaves were generally higher than those in roots, stems and fruits [11]. As reported by Santos et al. [44], spraying [Fe(MPP)3] iron fertilizer increased ferritin gene expression 2-fold, and the chlorophyll content and active iron content of soybean leaves were significantly increased by 29% and 36%, respectively. The results of this study indicated that spraying NCFe on IDC leaves could effectively improve the relative expression of the ferritin gene in leaves and then increase the active iron content in leaves.
The results of NCFe-regulated PME gene expression are shown in Fig. 8. After 72 h of treatment, the PME gene expression levels of PbPME1, PbPME3, and PbPME4 in CK plants were significantly higher than those in T1 and T3 plants, and the relative expression levels of PbPME1 and PbPME4 in T1 plants were significantly higher than those in T3 plants. However, there was no significant difference in the relative expression of PbPME3 between T1 and T3 plants. The relative expression levels of PbPME1, PbPME3, and PbPME4 in CK plants were 2.7, 1.8, and 7.3 times those in T3 plants and 1.4, 1.8, and 1.7 times those in T1 plants, respectively. The results showed that compared with that in CK and T1 plants, the relative expression of PbPME1 and PbPME4 in plants sprayed with NCFe decreased the most, indicating that pectin will be in a more methylated form and not bind to iron.
Pectin is one of the main components of the cell wall [45] and is usually secreted from the Golgi apparatus into the cell wall in highly methylated forms [46] and then undergoes demethylation by PME, consequently increasing metal ion binding sites in the cell wall [47]. On the other hand, soil bicarbonate can increase the apoplast pH of leaves, and the activity of PME increases as the pH increases, thereby enhancing iron precipitation in the apoplast and reducing its bioavailability [48, 49]. In this study, NCFe could significantly reduce the expression of PbPME due to sulfonic acid groups on NCFe and effectively reduce the apoplast pH. A relatively PME activity further reduces the precipitation of iron in the apoplast and improves its bioavailability.
Overall, to prevent Fe chlorosis, the application of synthetic Fe chelates is a common practice to increase the solubility of Fe and function as a transporter through solution to the plant [50]. However, the efficiency of foliar fertilization in pear Fe deficiency disease control was not significant as expected [10]. It is possible that leaf structure significantly affects the efficiency of Fe penetration and relocation when ferrous sulfate is sprayed on pear leaves [6, 8]. Nevertheless, the ferrous sulfate only acted on the adherent part, new leaves were still yellow because Fe was quickly fixed, and green spotting was still shown on the leaves [51]. Here, our results showed even re-greening in Fe-deficient leaves after NCFe treatment. This demonstrated that NCFe facilitated photosynthesis by stimulating the absorption of Fe in Fe-deficient leaves. The simplest possible mechanism to make Fe available is through inducing ferritin gene expression to express more ferritin for iron storage, down regulating PME gene expression and decreasing the binding affinity to iron on the cell wall but increasing the active Fe content in leaves. NCs had a strong ability to improve the dispersibility of active Fe when NCFe was applied in situ.