Foliar Application of Flavonoids (rutin) Regulates Phytoremediation Eciency of Amaranthus Hypochondriacus by Altering the Permeability of Cell Membranes and Immobilizing Excess Cd in the Cell Wall

The gap between the current serious soil heavy metal (HM) contamination situation and the low 22 efficiency of soil remediation has become one of the factors limiting economic development and human 23 health. The aim of this study was to propose a method to improve the efficiency of phytoremediation by 24 exogenous rutin application and to explain the potential mechanism. A series of rutin treatments were 25 designed to evaluate the biomass, cadmium (Cd) accumulation and phytoremediation efficiency 26 responses of Amaranthus hypochondriacus to different levels of rutin (0.5, 1.5, and 5 ppm) under 27 different Cd stress levels (10, 25, 50, and 100 ppm). The determination of cell membrane damage 28 indicators, the subcellular distribution of Cd and the establishment of a predictive model for Cd 29 accumulation were also carried out. The results showed a decline in cell membrane damage with rutin 30 application, and more Cd ions were immobilized in the cell wall than in the vacuole, resulting in an 31 increase in Cd tolerance in plants. The addition of rutin caused significant effects on the synthesis of 32 glutathione (GSH), including the advancement of the conversion of GSH to phytochelatins (PCs). Among 33 them, PC 2 and PC 3 in the leaves contributed the most to the high accumulation of Cd in Amaranthus 34 hypochondriacus according to the prediction model. Overall, the phytoremediation efficiency and 35 phytoextraction amount of Amaranthus hypochondriacus with foliar rutin application were improved 36 significantly by 260% and 319%, respectively. These findings can contribute to the further development 37 of soil remediation in Cd-contaminated fields. the application the to and the fixation


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Currently, with the rapid development of cities and industries, soil heavy metal (HM) pollution has 76 become a major problem affecting agricultural production, food safety, human health and environmental 77 safety ). Compared to traditional chemical and physical remediation 78 methods, phytoremediation is considered to be the most promising, cost-effective and environmentally 79 friendly method of soil decontamination (Liu et al. 2013, Qin et al. 2021). However, the shortcomings of 80 the low remediation efficiency of this method have become the main constraint to its wider application. The experimental soil (total Cd: 0.21 mg·kg -1 , available Cd: 0.06 mg·kg -1 , pH = 5.86) was collected from 117 the farmland of Mianzhu City, Sichuan Province, China. Cd treatment was carried out by uniformly 118 injecting of CdCl2•2.5H2O solutions into the soil of each pot to the designed soil Cd concentration of 10, 119 25, 50, and 100 mg·kg -1 . Seedlings of A. hypochondriacus were transplanted into experimental pots (80 120 cm × 40 cm × 40 cm) with 70 kg of soil after two weeks of soil equilibrium. Ten days after transplanting, 121 seedlings were fertilized with 1.2 g of liquid urea per pot. Exogenous rutin was applied 5 d after Cd 122 application during branching stage (the seedlings grew to 40 cm in height). The treatments of exogenous 123 rutin under different Cd conditions were as follows: R0, 0 mg·mL -1 ; R0.1, 0.1 mg·mL -1 ; R0.5, 0.5 124 mg·mL -1 ; R1.5, 1.5 mg·mL -1 ; and R5, 5 mg·mL -1 . The rutin solution was sprayed on leaf surfaces and 125 leaf backs until dripping. Three plants were randomly collected under each treatment condition after 3 d. 126 The plants were divided into three parts, namely, the roots, stems and leaves, for fresh and dried biomass 127 measurement. For each condition, triplicate analyses were performed. 128

Analysis of malondialdehyde, electrolyte leakage and cell vitality
129 Malondialdehyde (MDA) was determined to be a biomarker of lipid peroxidation and was measured to 130 investigate the impact of rutin on plant cell membranes. A 0.1 g fresh sample was homogenized in 5 mL 131 of 5% trichloroacetic acid (TCA). The cell suspension was centrifuged at 3000 rpm for 10 min. Then, 2 132 mL of 0.67% thiobarbituric acid (TBA) was added to the 2 mL supernatant and kept in a 100 °C water 133 bath for 30 min. After cooling, the mixture was centrifuged again at 3000 rpm for 10 min. The absorbance 134 of the supernatant was measured at 450 nm, 532 nm, and 600 nm. 135 The loss of cell membrane integrity in root, stem, and leaf tissues was estimated with electrolyte 136 leakage (EL) assessed by a conductivity meter. A 1.0 g sample was transferred into 20 mL of deionized 137 water and kept for 1 h at room temperature, and the conductivity of the solution was recorded as E 0 . 138 Then, the mixture was kept in a 100 °C water bath for 15 min, and the conductivity was marked as E 1 . 139 EL (%) was calculated by the ratio of E 0 to E 1 . 140 Cell relative vitality (CRV) (same for cell death) was determined by using the Evans Blue method 141 was set as 100% cell death (C 100% ). CRV was calculated as follows: 146 (1)

Measurement of Cd content
147 Dried plant samples were ground into powder by a grinding instrument. Then, 0.1 g samples from root, 148 stem and leaf parts were placed in a polytetrafluoroethylene crucible. Each sample was soaked for one 149 night in 10 mL of mixed acid (HNO3:HClO4 = 9:1). Then, the samples were digested on an electric hot 150 plate until nearly dry. After brining the volume to 10 mL with 1% HNO3 and filtration through a 0.45 μm 151 membrane filter, the samples were measured by inductively coupled plasma mass spectrometry. Collection and aggregation of raw data was conducted using EXCEL, and figures were generated with 181 GRAPHPAD PRISM8 and HIPLOT (www.hiplot.com). All data were statistically analyzed using SPSS 182 24, and significant differences between variables were determined using one-way analysis with the least 183 significant difference (LSD) post hoc test. Differences were statistically significant when p<0.05. 184 and 100 mg·kg -1 ), the biomass of roots tended to stabilize, and the accretion of total biomass mainly 204 relied on the stems and leaves. It was noted that almost no conspicuous toxicity symptoms of Cd were 205 found in the biomass accumulation of A. hypochondriacus with exogenous rutin application. Furthermore, 206 the biological dry matter in the 10, 25, 50, and 100 mg·kg -1 Cd treatments with rutin spray was 1.41-207 1.49, 1.07-1.59, 1.10-1.23, and 1.22-1.36 times higher than in the non-Cd-rutin control group, 208

Results
respectively. For maximal biomass results in this study, a higher concentration of rutin spraying was 209 optimum under medium-low Cd stress (10, 25, and 50 mg·kg -1 Cd application), while under higher Cd 210 stress (100 mg·kg -1 ), relatively low rutin application was sufficient.

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The Cd concentrations in roots, stems, and leaves were significantly changed with the application of rutin. 220 According to Fig. 2 (a) Cd concentrations were 1.56 (Cd 50-R5) and 1.29 (Cd 100-R1.5) times higher than those in the R0 group. 236 As shown in Fig. 2 (c), only the R1.5 and R5 treatments under 25 mg·kg -1 Cd application reported 237 significantly higher root Cd concentrations than the R0 group among all conditions under relatively low 238 Cd stress (10 and 25 mg·kg -1 ). Under relatively high Cd stress (50 and 100 mg·kg -1 ), the rutin treatment 239 groups still had a higher root Cd concentration.  The results of the BCF and PER are summarized in Table 1. Rutin application at each concentration 250 (R0.5, R1.5, and R5) increased the BCF of A. hypochondriacus for the treatments with all Cd stresses. 251 Especially in the R5 group, the BCF varied from 3.678, 3.366, 4.798, and 3.862 and was 1.46-2.22 times 252 higher than that in the R0 treatment. The presence of rutin also had a significant effect on the PER of A. 253 hypochondriacus. The highest PER (0.18%) was found in the Cd 25-R1.5 group, and other treatments 254 followed the trend that an increase in rutin concentration caused a higher PER.  intervention. In relation to that in the R0 group, EL in the roots and stems of A. hypochondriacus was 275 decreased by 14.15% and 22.73% in the R1.5 treatment, respectively. In terms of leaves, the EL dropped 276 by 7.12%, 20.05%, and 8.36% in the R0.5, R1.5 and R5 groups compared to the R0 group under 10 277 mg·kg -1 Cd conditions, respectively, and 6.12%, 20.73%, and 7.53% under 100 mg·kg -1 Cd conditions, 278 respectively. The CRV in the rutin treatments was higher than or equal to that in the control group (R0) 279 (Fig 3 (g)-(i)). The peak CRV value occurred in leaves under Cd 10-R5 conditions and was 1.28 times 280 higher than that of the control. Furthermore, MDA, EL, and CRV were significantly affected by rutin, 281 which changed the physiological parameters of the plants. and roots (Fig. 4 (b)-(c)), and the proportion of Cd in F CW in the R0.5 and R5 groups was higher than 300 in the R0 group. However, there were no substantial changes in the proportion of Cd in the F CW in stems 301 with or without rutin applied under 100 mg·kg -1 Cd stress. In contrast, the subcellular distribution of Cd 302 in roots (Fig. 4 (c)) changed with the same rule in accordance with leaves.

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Rutin was only detected in the leaves of A. hypochondriacus and revealed no significant alterations in 311 response to Cd stress (Fig. 5 (a)). Under the same level of Cd stress, exogenous rutin caused a rise in 312 endogenous rutin concentration. However, the rutin concentration in the R5 group was not much higher 313 than that in the R1.5 group, and even occasionally, it was lower than that in the R1.5 group. Moreover, 314 there was a surprising outcome in Fig. 5 (b)  The observed result was also somewhat counterintuitive: A. hypochondriacus cultivated solely with Cd 320 displayed a declining trend in GSH concentration with increasing Cd stress (Fig. 5 (c)). However, the 321 concentration of GSH increased drastically with the application of rutin, especially in the R5 group, where Cd wp , Cd L , Cd S , and Cd R are the concentration of Cd in whole plant, leaves, stems, and roots, 348 respectively (mg·kg -1 DW); Cd soil is the Cd concentration in soil (designed value, mg·kg -1 ); R en is the 349 endogenous rutin concentration in plants (mg·kg -1 ); PC 2R is the PC2 concentration in roots; and PC 2L 350 and PC 3L are the PC2 and PC3 concentration in leaves, respectively, with p < 0.001 in the T-test of each 351 coefficient.  This observation is also in line with a previous observation that rutin can enhance Cd uptake from soil to 362 plants. The PC2 concentration also indicated that the chelation and fixation of Cd in roots was also the 363 key to increasing Cd accumulation. Additionally, PCs in the leaves play a crucial role in the model of Cd 364 accumulation in leaves, stems, and roots, especially in leaves and roots, and PC3 is primarily responsible 365 for influencing Cd accumulation. The results showed that Cd content in all plant parts was highly 366 correlated with PCs in leaves, which may be due to the application of rutin leading to the transfer of the 367 hypochondriacus cultivated with rutin showed a lower MDA level in roots, stems, and leaves (Fig. 3), Since rutin can only be detected in the leaves, it is unknown whether the addition of rutin has a 389 direct effect on the roots and stems. However, due to the increased protective effect of rutin on the cell 390 membrane in leaves, the tolerance of photosynthetic plant organs to Cd is elevated, which promotes this 391 advantage in roots and stems at the same time.  concentration in A. hypochondriacus ( Fig. 1 and Fig. 2). However, the mechanism of function of rutin is 453 It is therefore evident that exogenous rutin actively functions in eliminating morphological retardation 458 and cell damage in A. hypochondriacus subjected to Cd stress. The increase in PER and BCF (Table 1)  determined that PC2 and PC3 in leaves are most relevant for PCs-driven Cd retention. In summary, this 475 study is the first to propose the application of endogenous phytoflavonoids to exogenous foliar sprays to 476 achieve higher Cd phytoextraction amount. Furthermore, these findings provide insights into a less costly 477 way to improve the low efficiency of phytoremediation. 478