Abstract
Nanocrystal cellulose possesses a strong capability to chelate Fe due to its adsorptive properties. Iron deficiency chlorosis (IDC) is a mineral disorder that remarkably weakens pear photosynthesis, causing declines in plant yields and quality. Conventional methods for controlling IDC generally lack efficiency and overuse chemicals. Foliar application of nanocellulose (NC)-Fe chelate (NCFe) provides a new approach to remediate IDC in pear (Pyrus betulifolia). In this study, NC was prepared by acidic hydrolysis using 64 wt% H2SO4 at 45 °C for 45 minutes. NCFe was formulated based on the net charge density of NC and ferrous sulfate (FeSO4) solution. The nanoparticle properties were characterized by transmission electron microscopy (TEM), dynamic light scattering, and conductometry. Pyrus betulifolia seedlings were pre-etiolated in an improved Hoagland’s nutrient solution and treated with bicarbonate. Changes in chlorophyll content, active Fe content, and photosynthesis rate in NCFe-treated leaves were determined by SPAD values, spectrophotometry, and photosynthetic apparatus, respectively. Ferritin genes (PbFER) and pectin methylesterase genes (PbPME) were extracted from leaf tissue, and gene expression profiles were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). The results showed that NCFe particles maintained a whisker-like morphology; the Z-average hydrodynamic diameter and zeta potential of NCFe measured by dynamic light scattering were 107.4 ± 3.0 nm and −9.7 ± 0.4 mV, respectively. When NCFe was prepared at a mixing ratio of 1:3000, the total chlorophyll content, active Fe content, and net photosynthetic rate of plant leaves were significantly enhanced by 23.8%, 65.9%, and 40.4% after 72 hours of treatment, respectively, compared with FeSO4 spraying. Importantly, NCFe treatment also significantly downregulated the expression of PbPME and upregulated the expression of PbFER, which are key genes regulating the active Fe content.
Iron (Fe) plays an important role in several basic physiological functions and is an important factor involved in pear (Pyrus spp.) tree growth and development. Iron deficiency chlorosis (IDC) is a worldwide problem that began in the 1930s, particularly in semiarid regions containing calcareous soils (Chapman, 1931). Since then, IDC has been reported in many varieties of oriental and occidental pear in different regions (El-Deen et al., 2018; He et al., 2013; Ikinci et al., 2016; Thomasraese and Staiff, 1988). IDC in pear trees decreases the leaf chlorophyll content, active Fe content, and photosynthetic capacity as well as the yield and quality of pear products (Álvarez-Fernández et al., 2011; Fernández et al., 2008). A previous study demonstrated that leaf nutrient absorption is usually quicker and more efficient than in the roots, especially at a high soil pH and calcium carbonate content (El-Dahshouri et al., 2017). Foliar applications of FeSO4, Fe ethylenediaminetetraacetic acid (Fe-EDTA), or Fe–ethylene diamine-N,N′-bis (hydroxy phenyl) acetic acid are conventional methods used to correct IDC in pear trees (El-Dahshouri et al., 2017). However, synthetic Fe-EDTA and Fe–ethylene diamine-N,N′-bis (hydroxy phenyl) acetic acid are expensive and contain large amounts of potential pollutants, which risk environmental safety (Yuan and Vanbriesen, 2006). In calcareous soil, the pH of plant leaf apoplasts is relatively high, which promotes ferrous Fe oxidation and fixation (Fernández and Ebert, 2005). Although the cost of spraying FeSO4 is relatively low, Fe(II) salts are rapidly oxidized after exposure to ambient air and cannot fully control IDC in pear trees (Álvarez-Fernández et al., 2004). Therefore, it is necessary to develop new and environmentally friendly Fe-chelating agents to improve the absorption of foliar-applied Fe or enhance its translocation efficiency from source to sink tissue (Malhotra et al., 2020).
The integration of nanotechnology and biotechnology has allowed for the development of new solutions for agriculturally sustainable applications. In recent years, growing interest has focused on nanofertilizers and nanopesticides in the agriculture and food sectors (Chhipa, 2017; Khan and Rizvi, 2017). Cellulose is one of the most abundant materials found in nature, and nanocellulose (NC) has recently spurred on scientific and economic interest in the research community. The advanced characteristics of NC are attributed to its small size, high surface area, and polyionic mechanical stiffness. Previous studies have shown that wood cellulose–based polyelectrolyte nanocomplexes can be used as novel carriers for protein delivery systems in agriculture (Shen et al., 2016; Song et al., 2012) and in environmental remediation as bioadsorbents (Bossa et al., 2017; Faiz Norrrahim et al., 2021). Recently, Baruah et al. (2020) investigated NC-Fe oxide nanobiocomposites in the remediation of contaminated groundwater and found that this nanocomposite possessed strong adsorptive properties and magnetic recoverability. However, as a comprehensive bionanomaterial, NC-Fe chelate (NCFe) has not yet been systematically studied in agriculture. The objective of this study was to investigate the effectiveness of NCFe in promoting Fe uptake, Fe activation, and photosynthesis to remediate IDC in pear, as well as to study the underlying molecular mechanism. The findings of this study may serve as a reference for Fe-chelating applications in plant IDC management.
Materials and Methods
Preparation of NC.
Cellulose, in the form of dissolving-grade softwood sulfite pulp (Temalfa 95A), was provided by the Rayonier Advanced Materials Company (Temiscaming, Québec, Canada). NC suspensions were prepared by acidic hydrolysis following previously described methods with minor modifications (Dong et al., 2016). Briefly, 20 g cellulose powder was milled in a universal grinder (FW100; Taisite Instrument Co., Tianjin, China) and then hydrolyzed in 200 mL 64 wt% H2SO4 at 45 °C for 45 min while stirring. Then, 500 mL precooled deionized (DI) water (4 °C), which was prepared with a Millipore Direct-Q 5 ultrapure water system (Merck KGaA, Darmstadt, Germany), was added to stop the reaction. The pellet was collected by centrifugation at 9000 gn and 4 °C for 15 min. The reaction was repeated three times under the same hydrolysis conditions. The final product was washed three times and redispersed in 200 mL DI water and dialyzed against DI water in dialysis tubing (JMD45-12∼14–0.5; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) to remove the acid residue until the pH of the dialysis water remained constant (pH, 5.4 ± 0.3).
The obtained suspension after dialysis was sonicated in an ice bath for 25 min (5 s/5 s) at 35% output with an ultrasonic processor (JY98-IIIN; Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China) to break down the aggregates. The stock suspension was stored at 4 °C for future use.
Characterization of NC.
The concentration of the NC product was determined from triplicate measurements of the weight difference of a 3-mL aliquot before and after drying for 2 h in an oven at 80 °C. The net charge densities of the NC suspension and ferrous sulfate (FeSO4) solution were determined from triplicate measurements after conductometric titration using a S470-USP/EP pH/conductivity meter with a conductivity probe (In Laboratory 730; Mettler Toledo International Co., Ltd., Zurich, Switzerland) following previously described methods (Wang and Roman, 2011). Briefly, 25 mL NC stock suspension was titrated with 0.02 M NaOH in 100-μL increments while stirring. Before titration, the ionic strength of the sample was adjusted to 0.01 mm using 5 M NaCl. The interval pH and conductivity values of the sample were recorded when the meter showed a stable reading. Based on the equivalence points of each range of the conductivity plot, the net charge density was calculated from the consumed titrant volume to neutralize counterions in the system.
The morphology of NC particles were observed using high-resolution transmission electron microscopy (TEM) instrument (JEM-2100; JEOL, Ltd., Tokyo, Japan). NC or NCFe with a concentration of 0.003% (w/v) was prepared by diluting the stock suspension with DI water. Samples were stained with saturated uranyl acetate negative stain for high-resolution imaging.
The Z-average hydrodynamic diameter (cumulant mean) and zeta potential of the NC nanoparticles were determined by dynamic light scattering following previously described methods (Wang and Roman, 2011). Measurements were performed in triplicate at 25 °C using Brookhaven Instruments with Zeta Plus Particle Sizing Software (NanoBrook Omni; Brookhaven Instruments Corp., Holtsville, NY). Samples were diluted in stock solution without adjusting the pH or ionic strength.
NCFe formulation and characterization.
NCFe was prepared by mixing NC with FeSO4 solution based on the net charge density. Based on our pretests, three formulations of NCFe with charge density NC:Fe ratios of 1:300, 1:3000, and 1:30,000 were prepared (Table 1). FeSO4 and Fe-EDTA are commonly used in 2 mmol⋅L−1 doses and were used as positive controls, and DI water served as the negative control. A supplement of 0.15% Tween-80 was used to increase the adsorption capacity. NCFe nanoparticles were characterized using the same methods for NC characterization.
Formulations of different experimental treatments.
Pre-etiolated seedlings of Pyrus betulifolia. P. betulifolia
Bunge seeds were used as the plant material. Samples were stratified in river sand at a weight ratio of 1:10 and incubated in a refrigerator at 4 °C for 25 d. The germinated seeds were planted in a soil matrix with 3 peat: 1 perlite: 1 vermiculite (by volume) and grown in a greenhouse. After six to eight leaves emerged, seedlings were transferred to a hydroponic pot containing improved Hoagland’s nutrient solution [6 mm KNO3, 4 mm Ca(NO3)2·4H2O, 2 mm KH2PO4, 1 mm MgSO4·7H2O, 50 μM KCl, 25 μM H3BO3, 2 μM MnSO4·H2O, 2 μM ZnSO4·7H2O, 0.5 μM CuSO4·5H2O, 0.5 μM H2MoO4 (85% MoO3), and 100 μM Fe(III)-EDTA]. After 1 week, plants were subjected to bicarbonate treatment [0.01 μM Fe(III)-EDTA, 2 mm NaHCO3, and 0.5 g⋅L−1 CaCO3; pH 7.2] for 1 month to cultivate IDC pear seedlings with three seedlings per pot and 15 min of ventilation per hour in a greenhouse (Guo et al., 2017).
When plants grew to 30 cm in height, seedlings were moved to a growth chamber under a 16/8 h light/dark photoperiod (26 °C/18 °C). After Fe deficiency treatment, the chlorophyll (a + b) content of the leaves was measured by a SPAD-502 portable chlorophyll analyzer (Konica Minolta Investment Ltd., Shanghai, China). Leaves with SPAD values ranging from 10 to 30 were labeled for further measurements. The mean SPAD value of each pot was calculated using 20 leaves.
Seedling treatments.
Experimental treatments were performed by foliar applications on pear seedling leaves. During the experiment, NCFe samples were freshly prepared by adding FeSO4 to NC before spraying. Then, three plants in each pot were evenly sprayed on the leaves with 8 mL solution (Table 1). The final dose of Fe was 2 mmol⋅L−1 in all treatments. The experiments were carried out in triplicate.
The root area was covered with plastic film to avoid potential contamination by the nutrient solution in the form of liquid from the leaves. After the liquid on the leaves dried, treated plants were transferred to a growth chamber. The phenotypes of the treated plants were observed, and related physiological indexes were determined after 72 h. The leaves were separated, put in liquid nitrogen, and stored at −80 °C in a refrigerator for RNA extraction.
Physiological parameters and indicator measurements.
The SPAD values of leaves were measured using a SPAD-502 portable chlorophyll analyzer (Konica Minolta Investment Ltd., Shanghai, China), and each leaf was measured three times. The middle part of the leaves was measured to avoid the midrib area.
To determine the active Fe content, the leaves were cut off from the plants without petioles. After washing, the leaves were dried at 105 °C for 30 min, followed by 70 °C for 1 week, in a blast drying oven (DHG-9070A; Shanghai Huitai Equipment Manufacturing Co., Ltd., Shanghai, China) until reaching a constant weight. Measurements were conducted following previously described methods (Zhai et al., 2016). Briefly, 100 mg dried leaves were crushed and extracted with 10 mL 1.0 mol⋅L−1 hydrochloric acid for 24 h while shaking. After filtration, the active Fe content in the extract solution was determined using an atomic absorption spectrophotometer (ZEEnit-700P; Analytik Jena AG, Jena, Germany).
The photosynthesis parameters of the leaves, including net photosynthetic rate (Pn), stomatal conductance (gS), intercellular CO2 concentration (Ci), transpiration rate (Tr), saturated vapor pressure difference (VPD), and water use efficiency (WUE), were measured using a portable photosynthetic apparatus (CIRAS-3; PP Systems, Amesbury, MA).
Analysis of active Fe-related gene expression.
For ferritin and pectin methylesterase (PME) extraction and analysis, leaf tissues were individually pulverized thoroughly with a mechanical grinding machine (KZ-III-FP; Servicebio, Wuhan, China) into a fine powder. Total RNA was extracted using an RNApure plant kit (CW0559; CWBio, Beijing, China) following the manufacturer’s instructions. RNA quality and quantity were checked using a spectrophotometer (Nanodrop 2000; Thermo Fisher Scientific Inc., Wilmington, DE). Single-stranded cDNA was synthesized using a ReverTra Ace qPCR RT kit (FSQ-101; Toyobo, Shanghai, China) following the manufacturer’s instructions. Primer sequences were designed using Primer3 online software (Table 2).
PCR primers for the ferritin and pectin methylesterase genes.
qRT-PCR was performed on a TL988 real-time PCR system (Tianlong, Xi’an, China) using 2X Universal SYBR Green Fast qPCR mix (RK21203; Abclonal, Suzhou, China) under the following reaction conditions: 95 °C denaturation for 5 min, 40 cycles for 5 s at 95 °C and 34 s at 60 °C, followed by melt curve stages to check that only single products were amplified. Every qRT-PCR assay was performed in biological triplicate. PbACTIN was used as the reference gene for normalizing the templates. Expression profiles were analyzed using the 2−ΔΔCT method (Schmittgen and Livak, 2008).
Statistical analyses.
All treatments were carried out in triplicate. The samples were collected randomly from the treatments, and each treatment had three measurements. Results are presented as mean ± sd. SPSS v22 (IBM Corp., Armonk, NY, USA) was used for the statistical analyses. Sample means were compared using analysis of variance. Mean separation was determined by Duncan’s multiple range test, with significance set at P < 0.05.
Results
Physicochemical properties of NC and NCFe.
The morphologies of NC and NCFe particles were visualized by TEM. NC particles had rod-like whiskers (Fig. 1A), which agreed with previous reports on NC (Beck-Candanedo et al., 2005; Wang and Roman, 2011). When NC was 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 dynamic light scattering were 84.3 ± 0.2 nm and −47.3 ± 1.7 mV, respectively, with a polydispersity index of 0.20 ± 0.01 (Fig. 2A and B). The particle size and zeta potential of NCFe were 107.4 ± 3.0 nm and −9.7 ± 0.4 mV, respectively, with a polydispersity index of 0.36 ± 0.05 (Fig. 2C and D). A typical V-shaped titration curve of the NC conductivity is shown in Fig. 3. The net charge density calculated from the consumption of titrant for sulfate group neutralization was 112 ± 6.4 mmol⋅kg−1.
Transmission electron microscopy images of nanocellulose (NC) and NC-Fe chelate (NCFe) particles. A solution of (A) 0.03% (w/v) NC or (B) 0.03% (w/v) NCFe was prepared by diluting the stock suspension with deionized water.
Citation: HortScience 57, 4; 10.21273/HORTSCI16404-21
Transmission electron microscopy images of nanocellulose (NC) and NC-Fe chelate (NCFe) particles. A solution of (A) 0.03% (w/v) NC or (B) 0.03% (w/v) NCFe was prepared by diluting the stock suspension with deionized water.
Citation: HortScience 57, 4; 10.21273/HORTSCI16404-21
Transmission electron microscopy images of nanocellulose (NC) and NC-Fe chelate (NCFe) particles. A solution of (A) 0.03% (w/v) NC or (B) 0.03% (w/v) NCFe was prepared by diluting the stock suspension with deionized water.
Citation: HortScience 57, 4; 10.21273/HORTSCI16404-21
Particle size and zeta potential of nanocellulose (NC) and NC-Fe chelate (NCFe) particles measured by dynamic light scattering. (A) Size and size distribution of NC (0.03% w/v). (B) Zeta potential of the NC suspension. (C) Size and size distribution of NCFe formulated at a charge density ratio of 1:3000. (D) Zeta potential of NCFe with the same formulation.
Citation: HortScience 57, 4; 10.21273/HORTSCI16404-21
Particle size and zeta potential of nanocellulose (NC) and NC-Fe chelate (NCFe) particles measured by dynamic light scattering. (A) Size and size distribution of NC (0.03% w/v). (B) Zeta potential of the NC suspension. (C) Size and size distribution of NCFe formulated at a charge density ratio of 1:3000. (D) Zeta potential of NCFe with the same formulation.
Citation: HortScience 57, 4; 10.21273/HORTSCI16404-21
Particle size and zeta potential of nanocellulose (NC) and NC-Fe chelate (NCFe) particles measured by dynamic light scattering. (A) Size and size distribution of NC (0.03% w/v). (B) Zeta potential of the NC suspension. (C) Size and size distribution of NCFe formulated at a charge density ratio of 1:3000. (D) Zeta potential of NCFe with the same formulation.
Citation: HortScience 57, 4; 10.21273/HORTSCI16404-21
Conductometric titration curves of nanocellulose (NC). A total of 25 mL NC stock suspension was titrated with 0.02 m NaOH. The ionic strength of the solution was adjusted to 0.01 mm with NaCl. Results are presented as the mean of three measurements.
Citation: HortScience 57, 4; 10.21273/HORTSCI16404-21
Conductometric titration curves of nanocellulose (NC). A total of 25 mL NC stock suspension was titrated with 0.02 m NaOH. The ionic strength of the solution was adjusted to 0.01 mm with NaCl. Results are presented as the mean of three measurements.
Citation: HortScience 57, 4; 10.21273/HORTSCI16404-21
Conductometric titration curves of nanocellulose (NC). A total of 25 mL NC stock suspension was titrated with 0.02 m NaOH. The ionic strength of the solution was adjusted to 0.01 mm with NaCl. Results are presented as the mean of three measurements.
Citation: HortScience 57, 4; 10.21273/HORTSCI16404-21
Phenotypes of pear leaves treated with NCFe.
As shown in Fig. 4, after 72 h of NCFe (T2–T4) and Fe-EDTA (T5) treatment, a more evenly distributed green color appeared in the leaves, whereas FeSO4 (T1)-treated leaves only partially recovered with an unevenly distributed green color and the yellow leaves displayed green spots. Thus, it was concluded that increased Fe uptake occurred under the NCFe treatments.
Phenotypes of pear seedling leaves under different treatments. CK: deionized water; T1: 2 mmol⋅L−1 FeSO4; T2–T4: chelate obtained with a nanocellulose:Fe charge ratio of 1:300, 1:3000, and 1:30,000, respectively; T5: 2 mmol⋅L−1 Fe–ethylenediaminetetraacetic acid. The final concentration of Fe in the chelates was 2 mmol⋅L−1 FeSO4. Scale bar = 1 cm.
Citation: HortScience 57, 4; 10.21273/HORTSCI16404-21
Phenotypes of pear seedling leaves under different treatments. CK: deionized water; T1: 2 mmol⋅L−1 FeSO4; T2–T4: chelate obtained with a nanocellulose:Fe charge ratio of 1:300, 1:3000, and 1:30,000, respectively; T5: 2 mmol⋅L−1 Fe–ethylenediaminetetraacetic acid. The final concentration of Fe in the chelates was 2 mmol⋅L−1 FeSO4. Scale bar = 1 cm.
Citation: HortScience 57, 4; 10.21273/HORTSCI16404-21
Phenotypes of pear seedling leaves under different treatments. CK: deionized water; T1: 2 mmol⋅L−1 FeSO4; T2–T4: chelate obtained with a nanocellulose:Fe charge ratio of 1:300, 1:3000, and 1:30,000, respectively; T5: 2 mmol⋅L−1 Fe–ethylenediaminetetraacetic acid. The final concentration of Fe in the chelates was 2 mmol⋅L−1 FeSO4. Scale bar = 1 cm.
Citation: HortScience 57, 4; 10.21273/HORTSCI16404-21
Enrichment of chlorophyll and active Fe content by NCFe.
In this study, the SPAD values (chlorophyll meter readings) were used to evaluate the effects of NCFe on the total chlorophyll content in the leaves (Fig. 5). The results revealed that, after 72 h of treatment, the total chlorophyll content increased. Treatments were ordered from high to low as follows: T3 > T4 > T2 > T5 > T1 > CK. The total chlorophyll contents were enhanced in T2–T5 and were significantly higher than in T1 and CK. The SPAD values of T2–T5 significantly increased by 15.1%, 23.8%, 16.8%, and 13.9% when compared with T1 and significantly increased by 60.9%, 72.9%, 63.2%, and 59.2% when compared with CK, respectively. However, no significant differences were detected among T2–T4.
Chlorophyll contents of plant leaves after 72 h of treatment. CK: deionized (DI) water; T1: 2 mmol⋅L−1 FeSO4; T2–T4: chelates prepared with a nanocellulose:Fe charge ratio of 1:300, 1:3000, and 1:30,000, respectively; T5: 2 mmol⋅L−1 Fe–ethylenediaminetetraacetic acid. All suspensions were diluted with DI water. Different letters indicate significant differences (P < 0.05).
Citation: HortScience 57, 4; 10.21273/HORTSCI16404-21
Chlorophyll contents of plant leaves after 72 h of treatment. CK: deionized (DI) water; T1: 2 mmol⋅L−1 FeSO4; T2–T4: chelates prepared with a nanocellulose:Fe charge ratio of 1:300, 1:3000, and 1:30,000, respectively; T5: 2 mmol⋅L−1 Fe–ethylenediaminetetraacetic acid. All suspensions were diluted with DI water. Different letters indicate significant differences (P < 0.05).
Citation: HortScience 57, 4; 10.21273/HORTSCI16404-21
Chlorophyll contents of plant leaves after 72 h of treatment. CK: deionized (DI) water; T1: 2 mmol⋅L−1 FeSO4; T2–T4: chelates prepared with a nanocellulose:Fe charge ratio of 1:300, 1:3000, and 1:30,000, respectively; T5: 2 mmol⋅L−1 Fe–ethylenediaminetetraacetic acid. All suspensions were diluted with DI water. Different letters indicate significant differences (P < 0.05).
Citation: HortScience 57, 4; 10.21273/HORTSCI16404-21
The effects of NCFe on the active Fe content in the leaves are shown in Fig. 6. After 72 h of treatment, the active Fe content in T1–T5 significantly increased when compared with CK. More importantly, the active Fe content in the NCFe treatments (T2–T4) was much higher than in the FeSO4 (T1) or Fe-EDTA (T5) treatments. In particular, when the chelate was prepared with a NC:Fe charge ratio of 1:3000 (T3), the active Fe content of the leaves was significantly enhanced when compared with all other treatments. More specifically, the active Fe content of T3 increased by 800.8% when compared with CK and by 65.9% when compared with T1. These results demonstrated that NC had a strong ability to promote the active Fe content in the leaves, thereby controlling IDC in pear leaves, which was eventually reversed.
Active iron contents in the leaves after 72 h of treatment. CK: deionized water; T1: 2 mmol⋅L−1 FeSO4; T2–T4: chelates prepared with a nanocellulose:Fe charge ratio of 1:300, 1:3000, and 1:30,000, respectively; T5: 2 mmol⋅L−1 Fe–ethylenediaminetetraacetic acid. Different letters indicate significant differences (P < 0.05).
Citation: HortScience 57, 4; 10.21273/HORTSCI16404-21
Active iron contents in the leaves after 72 h of treatment. CK: deionized water; T1: 2 mmol⋅L−1 FeSO4; T2–T4: chelates prepared with a nanocellulose:Fe charge ratio of 1:300, 1:3000, and 1:30,000, respectively; T5: 2 mmol⋅L−1 Fe–ethylenediaminetetraacetic acid. Different letters indicate significant differences (P < 0.05).
Citation: HortScience 57, 4; 10.21273/HORTSCI16404-21
Active iron contents in the leaves after 72 h of treatment. CK: deionized water; T1: 2 mmol⋅L−1 FeSO4; T2–T4: chelates prepared with a nanocellulose:Fe charge ratio of 1:300, 1:3000, and 1:30,000, respectively; T5: 2 mmol⋅L−1 Fe–ethylenediaminetetraacetic acid. Different letters indicate significant differences (P < 0.05).
Citation: HortScience 57, 4; 10.21273/HORTSCI16404-21
Enhancement of Pn by NCFe.
To further explore the physiological mechanism of P. betulifolia treated with NCFe from a photosynthesis perspective, we mainly explored the effects of NCFe with an optimal NC:Fe ratio (T3) and compared this treatment with the photosynthetic parameters of the ferric sulfate (T1) and CK treatments, including the Pn, gS, Ci, Tr, VPD, and WUE of the leaves. As shown in Table 3, the Pn of T1 and T3 significantly increased after 72 h of treatment when compared with CK. The Pn of T3 was 121.7% higher than CK and 40.4% higher than T1. Additionally, the gS, Tr, and WUE of T3 significantly increased when compared with CK, but there were no significant differences in the gS or WUE between T1 and CK. The Ci and VPD of CK were significantly higher than T3, but no significant difference was detected between T3 and T1.
Effects of the experimental treatments on the photosynthetic parameters in pear leaves.
Regulation of relative gene expression.
We mainly studied the relative expression levels of P. betulifolia ferritin genes (PbFER1, PbFER2, PbFER3, and PbFER4) in this study. The results revealed that the expression levels of ferritin genes (PbFER1, PbFER2, and PbFER3) in T1 and T3 were significantly upregulated when compared with CK after 72 h of treatment. The relative expression levels of these three genes in T3 were significantly higher than in T1 (Fig. 7). The expression levels of PbFER1, PbFER2, and PbFER3 in T3 were 3.6-, 4.2-, and 4.0-fold greater than in CK and 1.5-, 1.6-, and 1.7-fold greater than in T1, respectively. The relative expression level of PbFER4 in T3 was significantly higher than in T1 and CK, but no significant difference was detected between T1 and CK.
Relative expression levels of ferritin family genes in pear leaves after 72 h of treatment. CK: deionized (DI) water; T1: 2 mmol⋅L−1 FeSO4; T3: chelate prepared at a nanocellulose:Fe charge ratio of 1:3000. All suspensions were diluted with DI water. Different letters indicate significant differences (P < 0.05).
Citation: HortScience 57, 4; 10.21273/HORTSCI16404-21
Relative expression levels of ferritin family genes in pear leaves after 72 h of treatment. CK: deionized (DI) water; T1: 2 mmol⋅L−1 FeSO4; T3: chelate prepared at a nanocellulose:Fe charge ratio of 1:3000. All suspensions were diluted with DI water. Different letters indicate significant differences (P < 0.05).
Citation: HortScience 57, 4; 10.21273/HORTSCI16404-21
Relative expression levels of ferritin family genes in pear leaves after 72 h of treatment. CK: deionized (DI) water; T1: 2 mmol⋅L−1 FeSO4; T3: chelate prepared at a nanocellulose:Fe charge ratio of 1:3000. All suspensions were diluted with DI water. Different letters indicate significant differences (P < 0.05).
Citation: HortScience 57, 4; 10.21273/HORTSCI16404-21
Relative expression levels of the pectin methylesterase genes in pear leaves after 72 h of treatment. CK: deionized (DI) water; T1: 2 mmol⋅L−1 FeSO4; T3: chelate prepared with a nanocellulose:Fe charge ratio of 1:3000. All suspensions were diluted with DI water. Different letters indicate significant differences (P < 0.05).
Citation: HortScience 57, 4; 10.21273/HORTSCI16404-21
Relative expression levels of the pectin methylesterase genes in pear leaves after 72 h of treatment. CK: deionized (DI) water; T1: 2 mmol⋅L−1 FeSO4; T3: chelate prepared with a nanocellulose:Fe charge ratio of 1:3000. All suspensions were diluted with DI water. Different letters indicate significant differences (P < 0.05).
Citation: HortScience 57, 4; 10.21273/HORTSCI16404-21
Relative expression levels of the pectin methylesterase genes in pear leaves after 72 h of treatment. CK: deionized (DI) water; T1: 2 mmol⋅L−1 FeSO4; T3: chelate prepared with a nanocellulose:Fe charge ratio of 1:3000. All suspensions were diluted with DI water. Different letters indicate significant differences (P < 0.05).
Citation: HortScience 57, 4; 10.21273/HORTSCI16404-21
The NCFe-regulated PME gene expression results are shown in Fig. 8. After 72 h of treatment, the PME gene expression levels of PbPME1, PbPME3, and PbPME4 in CK were significantly higher than T1 and T3, and the relative expression levels of PbPME1 and PbPME4 in T1 were significantly higher than in T3. However, there was no significant difference in the relative expression level of PbPME3 between T1 and T3. The relative expression levels of PbPME1, PbPME3, and PbPME4 in CK were 2.7-, 1.8-, and 7.3-fold greater than T3 and 1.4-, 1.8-, and 1.7- fold greater than T1, respectively.
Discussion
Physicochemical properties of NC and NCFe.
NC synthesis involves partial esterification using sulfuric acid under appropriate conditions (Wang and Roman, 2011). Hence, sulfonic groups on the surface of NC particles can be determined by conductometric titration (Raurich Sas, 1925; Usher, 1925). The surface chemistry of charged nanoparticles significantly affects their biological performance. The net charge density of NC in an aqueous suspension is an important parameter used to measure biological activity. A well-dispersed suspension can be easily applied on plant surfaces by the spraying method, and the size and size distribution of charged nanomaterials in an aqueous solution greatly affect the efficiency of spraying. A negative zeta potential value indicates that the nanoparticles are negatively charged. The net charge density of NC indicates the magnitude of ionizable groups on the surface of these particles. A higher net charge density or zeta potential of nanoparticles supplies a stronger repel force for suspending particles in a solution. In our study, these conditions provided the opportunity to chelate Fe ions for the transportation and dispersion of Fe into a biological system (Chaichi et al., 2017).
Effects of NCFe on the phenotypes of pear leaves.
The rod-like particles of NC, which had a negative zeta potential, indicated that the particles were negatively charged (Fig. 2B). It has been proven that NC has a higher adsorption capacity and better binding affinity than other similar materials at the microscale (Faiz Norrrahim et al., 2021). When NC is mixed with Fe2+, the high content of available sulfonic groups on the surface of NC particles is used as an anchor point for the simultaneous reduction and stabilization of NC-supported Fe2+. The use of NC as a carrier to restore the availability of Fe could be a valuable and sustainable strategy for reducing the effects of IDC in pears and other crops (Baldi et al., 2018). This phenomenon may be attributed to the increased interaction between the positively charged metal salt and negatively charged NC due to the presence of sulfonic groups on the NC surface and Fe chelating properties (Dhar et al., 2015; Kang et al., 2021).
Iron ions that bound to anionic NC whiskers increased the Fe transportation capability and promoted active Fe migration to Fe-deficient plants. According to a study conducted by Wang et al. (2016), ferrous sulfate (FeSO4·7H2O) could be chelated with microcrystalline cellulose and attapulgite, and the release of Fe was pH controlled in leaf cells when applied as a foliar fertilizer. Under acid conditions, the strength of ionization of nanocrystal cellulose was reduced and caused the chelated Fe(II) to become isolated, facilitating the release of Fe(II) from the chelate (Wang et al., 2011; Wang and Roman, 2011). In this study, the phenotype of pear leaves treated with ferrous sulfate alone indicated that ferrous ions may not be efficiently absorbed or evenly transported to other places (Fig. 4, T1). In the leaves treated with NCFe (Fig. 4, T2–T4), depending on the NC:Fe charge ratio, ferrous ions were easily transported into plant cells and increased the total chlorophyll and active Fe contents, thereby improving the Pn. We therefore assumed that NC particles performed as carriers to bring ferrous ions into the leaves and distributed them evenly; the leaves subsequently turned green more sufficiently. Comparably, the greenness of Fe-EDTA–treated plants (Fig. 4, T5) was between the FeSO4–treated and the NCFe-treated plants, indicating that the level of returning to a green color may be dependent on foliar fertilizer formulation and/or facilitators. This ferrous foliar fertilizer displayed obviously positive effects on the growth of corn (Zea mays L.), resulting in increased plant heights and chlorophyll contents in the leaves compared with FeSO4 spraying. We observed that NCFe prepared at a charge ratio of 1:3000 was the optimal formulation for controlling IDC. However, the release dynamics and metabolic mechanism of NCFe within Fe-deficient plants at the molecular level should be further explored in future studies.
Pn enhancement promoted by NCFe.
A previous study showed that the sulfate group density plays an important role in the chelation of Fe to NC and in their biological activity (Jiang et al., 2013). Our results indicated that different concentrations of NC in the NCFe formulation had different effects on IDC recovery (Fig. 4). At an appropriate chelation formulation, NCFe could enhance physiological indicators (Fig. 5) and increase the total chlorophyll contents up to 72.9% and 23.8% when compared with CK and T1, respectively (Fig. 4). A small increase in chlorophyll and carotenoids collectively form a reaction center where the absorbed light energy is initially converted into chemical energy, resulting in healthy plant growth (Hashimoto et al., 2016).
Photosynthesis is a complex, comprehensive photochemical and biochemical process (Tighe-Neira et al., 2018). It has been reported that metallic nanoparticles and oxides can be used as photocatalysts to convert light to energy and promote the Pn when associated with the structure and function of plant photosynthesis (Liu et al., 2017). Preventing and controlling IDC is difficult and often leads to poor results, but Cu-Fe chelation, for example, enhanced the Pn in IDC grape leaves (Ma et al., 2019). Our results also demonstrated that spraying NCFe significantly improved the photosynthetic capacity of leaves when compared with spraying FeSO4.
Molecular mechanism of NCFe in IDC recovery.
Ferritin plays an important role in Fe storage in plants. The relative expression levels of PbFER1, PbFER2, PbFER3, and PbFER4 in plant leaves were found to be generally higher than those in the roots, stems, or fruits (Xi et al., 2011). As reported by Santos et al. (2021), spraying Fe fertilizer (3-hydroxy-4-pyridinone Fe-chelate) increased ferritin gene expression by 2-fold, and the chlorophyll and active Fe content in soybean leaves significantly increased by 29% and 36%, respectively. Our results showed that the expression levels of ferritin genes (PbFER1, PbFER2, and PbFER3) in T1 and T3 were significantly upregulated when compared with CK after 72 h of treatment, and the relative expression levels of these three genes in T3 were significantly higher than in T1 (Fig. 7). These results indicated that spraying NCFe on IDC leaves effectively improved the relative expression of ferritin genes, thereby increasing the active Fe content.
Pectin is a main component of the cell wall (Cosgrove, 2005). It is usually secreted from the Golgi apparatus into the cell wall in highly methylated forms and undergoes demethylation by PME, consequently increasing metal ion binding sites in the cell wall (Gaffe et al., 1992; Zhang et al., 2011). Soil bicarbonate increases the apoplast pH in plant leaves, and the activity of PME increases as pH increases, thereby enhancing Fe precipitation in the apoplast and reducing its bioavailability (Del Corpo et al., 2020; Mengel et al., 1994). In this study, we demonstrated that NCFe significantly reduced the expression of PbPME, which may be due to the sulfonic acid groups on NCFe effectively reducing the apoplast pH, thereby further reducing PME activity and the precipitation of Fe in the apoplast, thus ultimately improving Fe bioavailability.
Conclusions
Cellulose is a renewable biomaterial. Its special physiochemical properties lend it a wide range of applications in many areas. Nanocrystal cellulose whiskers produced by acidic hydrolysis carry negative charges introduced from acids. Therefore, anionic nanocrystal cellulose has a promising capability to chelate Fe ions due to its adsorptive properties. This study demonstrated that NC:Fe chelated at a charge density ratio of 1:3000 was the optimal formulation to remediate pear IDC. This treatment significantly downregulated the expression of PbPME and upregulated the expression of PbFER, which increased the active Fe content. NCFe therefore strongly promoted chlorophyll content and increased the photosynthesis rate. This study will serve as a reference and complementary strategy for Fe-chelating applications in plant IDC management.
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