Protective Effect of foliar application of sulfur on photosynthesis and antioxidative defense system of rice under the stress of Cd

Journal Pre-proof Protective effect of foliar application of sulfur on photosynthesis and antioxidative defense system of rice under the stress of Cd

Jiahao Liu, Hong Hou, Long Zhao, Zaijin Sun, Hua Li PII:

S0048-9697(19)36226-6

DOI:

https://doi.org/10.1016/j.scitotenv.2019.136230

Reference:

STOTEN 136230

To appear in:

Science of the Total Environment

Received date:

8 August 2019

Revised date:

19 November 2019

Accepted date:

18 December 2019

Please cite this article as: J. Liu, H. Hou, L. Zhao, et al., Protective effect of foliar application of sulfur on photosynthesis and antioxidative defense system of rice under the stress of Cd, Science of the Total Environment (2019), https://doi.org/10.1016/ j.scitotenv.2019.136230

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© 2019 Published by Elsevier.

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Protective Effect of Foliar Application of Sulfur on Photosynthesis and Antioxidative Defense System of Rice under the Stress of Cd Jiahao Liua, b, Hong Houb, **, Long Zhaob, **, Zaijin Sunb, Hua Lic, * a

State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese

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Institute of Loess Plateau, Shanxi University, Taiyuan, Shanxi 030006, China

Research Academy of Environmental Sciences, Beijing 100012, China School of Environmental Science and Resources, Shanxi University, Taiyuan,

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Shanxi 030006, China

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* Corresponding author. ** Corresponding author.

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Abstract

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E-mail addresses: [email protected] (H. Li), [email protected] (H. Hou).

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This paper investigates the effect of foliar application of sulfur on photosynthesis and antioxidative defense system of rice under the stress of Cd. The initial field studies showed that foliar spray of S was effective for reducing Cd concentration in rice and increasing the grain yield. However, the physiological mechanisms remain less clear on how the foliar application of S alleviates Cd toxicity in rice. Chlorophyll fluorescence, as a measure of photosynthesis, was taken to estimate the efficiency of photosystem II (PSII) photochemistry after the foliar application of S. The increase of photosynthetic parameters, i.e. the maximum photochemical efficiency of PSII reaction center (Fv/Fm), the actual PSII photochemical efficiency (PSII), the 1

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photochemical quenching coefficient (qP), indicated that the foliar treatment alleviated the toxicity of Cd to PSII. The decrease of non-photochemical quenching coefficient (NPQ) indicated the increase of photochemical reaction efficiency with more absorbed light energy for photochemical reactions. Fourier Transform Infrared (FTIR) spectra showed that the foliar treatment stimulated the syntheses of lignin,

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lipids, aliphatic acid, polysaccharides, carboxylate and proteins. Micrographs of

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transmission electron microscope (TEM) also revealed the reduced mobility of Cd in

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cells. Foliar application of S effectively reduced the damage of Cd stress by

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maintaining the integrity of cell structure and participating in metabolic activities such

Keywords:

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as protein synthesis.

Foliar application; Cadmium; Sulfur; Photosynthesis; Chlorophyll

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1. Introduction

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fluorescence

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The loss of food production caused by heavy metal contamination in China is greater than 10,000,000 tons per year, resulting in a total economic loss of more than 20 billion yuan (Yuan et al., 2018). Among the well-known phytotoxic heavy metals in the environment, Cd has great harm due to its high water solubility, mobility, persistence, and toxicity even if it is in a small amount. Approximately 7.0% of the land area in China has been contaminated by Cd at different levels (Huang et al., 2017). Soil Cd pollution results in various problems such as inhibition of photosynthesis, alteration of ion homeostasis, poor quality of products, loss of yield, and metal toxicity to animals and humans. Rice is the most widely cultivated crop in 2

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the world, and the staple food for approximately 3 billion people (Huang et al., 2019). The symptoms of Cd toxicity in rice are associated with leaf chlorosis, growth inhibition and the disruption of key physiological processes including photosynthesis (Augustsson et al., 2018). Many measures have been taken to reduce the accumulation of Cd in rice, which

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include soil remediation engineering, agricultural practice and phytoremediation

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(Gustin et al., 2018). These remediation technologies are complicated and often

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expensive at the field scale. Alternative to or along with these measures, foliar

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yield (Sanglard et al., 2014).

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dressing of S maybe offer a potential to mitigate Cd stress in rice without decreasing

Sulfur has been proved to be useful for the growth and development of crop plants.

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Although the reported effects of S varied with the plant species, it has the ability to

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increase the tolerance in crop plants to different stresses (Li et al., 2017). Sulfur has

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been shown to improve disease resistance and light interception as well as photosynthesis, modify nutrient imbalance, minimize mineral toxicity and enhance abiotic tolerance (Cao et al., 2018; Fang et al., 2018). The deficiency of S could disturb the production of plant chlorophyll and the efficiency of protein synthesis (Duncan et al., 2018). Fatma et al. (2014) found the excess supplement of S improved photosynthesis and growth under salt stress via the increased production of glutathione (GSH) in mustard. The biogeochemical cycle of S can profoundly affect the heavy metals and their transformation in soils. It has been reported that the addition of S fertilizer led to the reduced mobility of Cu by the conversion of 3

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bioavailable Cu to Cu2S and Cu-cysteine in the rhizosphere soils (Sun et al., 2016). Zhang et al (2013) reported that the addition of S reduced Cd absorption, which was attributed to the synthesis of thiol pool like GSH, phytochelatins (PCs), and non-protein thiol (NPT) in rice plants. In terms of nutrition, the sufficient supply of S is required to maintain the optimum yield. For example, oilseed rape and Brassica

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species require S during their growth for the synthesis of both protein and naturally

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occurring glucosinolates (Zhao et al., 1993). Therefore, S is considered as a

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high-quality fertilizer that may be used for developing ecological agriculture.

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However, the availability of S fertilizer applied into soil is relatively low, which might

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be absorbed by organic matters and/or minerals in soils. Although foliage dressing has been widely used as nutrition fertilizer in agricultural

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practices for centuries (Reuveni and Reuveni, 1995; Bai et al., 2008; Augusto et al.,

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2018), it is just in recent years the foliage dressing has received attention for the

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mitigation of heavy metal pollution in edible plant parts. Foliar dressing could alleviate Cd stress in rice by improving photosynthesis, which plays a central role in plant biosynthesis, providing an interactive link between the internal metabolism of the plant and its external environment. The initial symptoms of environmental stress are clearly detectable due to the changes in photosynthesis. Measuring fluorescence of chlorophyll is a useful tool to identify structural damage to photosynthetic apparatus (De Faria et al., 2013). Photosynthetic parameters such as net photosynthetic rate (Pn), intercellular CO2 concentration (Ci), and stomatal conductance (Gs) were found reversely proportional to the concentration of Cd in rice (Gao et al., 2018). 4

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Antagonistic effects have also been reported to alleviate the toxicity of Cd because plants have evolved complicated mechanisms serving to limit the uptake, accumulation of toxic metals (Drzeżdżon et al., 2018). Foliar spraying of Se can trigger the activities of superoxide dismutase (SOD) and ascorbate peroxidase in the leaves of Brassica campestris to remove excess free radicals (Ding et al., 2017). The

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mechanism of foliar Si application may be related to the Cd sequestration in shoot cell

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walls (Liu et al., 2009).

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To our best knowledge, no one has studied the effect of foliar application of S on

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the resistance to Cd stress in rice. It was hypothesized that the foliar-applied S could

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regulate Cd in rice by reducing Cd uptake and improving the antioxidant defense system. However, how these reactions affect key physiological processes, such as

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photosynthesis, has not been fully clarified; and how foliar application affect Cd

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uptake at subcellular and molecular level have not been examined (Cao et al., 2014).

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This study was to examine the effect of foliar application of S on Cd in grain and rice yield, and its relationship with photosynthesis, chlorophyll fluorescence and antioxidative defense system. Possible protective role of S was also investigated using FTIR analysis and microscopic analysis.

2. Materials and methods 2.1 Experimental treatments 2.1.1 Field experiment Field trial was conducted in a paddy filed in Changsha County, Hunan, China (28°15′ N, 113°14′ E), in the subtropical region with a long history of double-rice 5

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cropping. The experiment soil was slightly contaminated with Cd according to the Environmental Quality Standards for Soils of China (GB-15618-2018). The available Cd in soil was relatively high at 0.44 ± 0.04 mg kg-1. Field experiments were conducted in both early and late rice seasons during 2017. The rice seeds were provided by the Hunan Academy of Agricultural Sciences. The cultivars of rice were

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Zhongzao39 and Yuzhenxiang for early and late rice, respectively. The experimental

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plot was laid out in randomized block design with three replications. The plot size

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was 15 m  10 m, including 1.0 m paths between the plots and 1.5 m discarded at

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each end to avoid cross impact. Rice seeds were sterilized with 5% sodium

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hypochlorite solution for 15 min before seedling nursery and rinsed with tap water. Soil fertilization and other management followed local rice conventional operating

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practices.

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Foliar S treatments were conducted at different stages to explore the optimal

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treatment time. The spray times were at the seedling stage (25-35d), tillering stage (60-70d), and flowering stage (80-90d) respectively. 0.5 g L−1 Na2S was supplied as the source of S based on our preliminary studies with the same rice cultivar (Liu et al., 2019). The estimation of applied S was 112.5 g per hectare. Foliar spray was done twice on leaves at the seedling stage, tillering stage or flowering stage, evenly wetting all leaves without dripping. The control groups were sprayed with the same amount of deionized water. Rice grain was harvested after maturity. Grain Cd concentration was determined using inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7500CX, USA) after being digested with HNO3: H2O2 (v:v = 3:1). Grain yield was 6

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measured according to the method of Dobermann and Fairhurst (2000).

2.1.2 Greenhouse experiment The greenhouse experiment was conducted at the Chinese Research Academy of Environmental Sciences, Beijing, in August, 2018. Rice seeds (cultivar Yuzhenxiang) were sterilized with 5% sodium hypochlorite solution for 15 min, rinsed thoroughly

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with deionized water, then all sterilized seeds were placed in quartz sand to germinate

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in an automatically controlled growth chamber (with a 14 h light period, day

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temperature of 28°C and night temperature of 20°C and relative humidity of 60%–

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70%) for 5 days. After germination, the seedlings were transferred into plate holes on

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plastic pot at a density of 20 seedlings per pot containing Kimura B nutrient solution, with 0.37 mM (NH4)2SO4, 0.18 mM KH2PO4, 0.18 mM KNO3, 0.55 mM MgSO4,

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0.09 mM K2SO4, 0.37 mM Ca (NO3)2, 50 μM Fe(II)-EDTA, 1 μM ZnSO4, 1 μM

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CuSO4, 5 μM MnSO4, 10 μM H3BO4, 0.2 μM CoSO4, 0.5 μM Na2MoO4, 100 μM

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NaCl. The pH of solution was maintained at 5.0-6.0 with 1 mM HCl or 1 mM NaOH solution. Nutrition solutions were renewed every 3 day. After 3 weeks, plants were exposed to Cd by adding CdCl2 to the nutrition solution at five different concentrations at 0.2, 0.5, 1.0, 2.0, 5.0 mg L-1. The foliar spray of S, as Na2S at 0.5 g L−1, was applied at the tillering stage with a hand-operated knapsack sprayer in the early morning. Five pairs of parallel treatments along with the control were as follows: (1) Control, 0 mg L-1 Cd stressed plants with foliar spray of distilled water; (2) 0.2 mg L-1 Cd stressed plants with foliar spray of distilled water and foliar spray with S; (3) 0.5 mg L-1 Cd stressed plants with foliar spray of distilled water and foliar spray with 7

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for further measurements.

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2.2 Photosynthetic parameters and Chlorophyll fluorescence

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measurements

Photosynthetic parameters were determined simultaneously via conducting

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measurements of chlorophyll fluorescence using two cross-calibrated portable

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open-flow gas exchange systems (LI-6400XT, LI-Cor Inc., Lincoln, NE, USA) equipped with integrated fluorescence chamber heads (LI-6400-40, Li-Cor Inc.). The

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atmospheric conditions of the leaf chamber were as follows: light intensity 1.0 mmol

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m-2 s-1, air flow rate 500 mmol s-1, leaf temperature 25 °C. After foliar treatments at

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the tillering stage, photosynthetic parameters were measured on flag leaves from 9:0012:00, assuming that the rice leaves were fully functional at that time to measure Pn, Gs, Ci, and transpiration rate (Tr). All measures were taken from the same leaf. Three replicates were randomly selected from three different plants, and each replicate from different leaves. Every parameter was measured five times at different spots on a single leaf, and recorded as the mean of five measurements. The 30 minutes dark-adapted leaf tissues were illuminated with weak modulated measuring beam (0.03 μmol m-2 s-1) to obtain the initial fluorescence (Fo). The saturated white light pulse of 8000 μmol m-2 s-1 photon was applied for 0.8 s to ensure maximum fluorescence emissions (Fm). Fo was the minimum fluorescence yield when the 8

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2.3 FTIR measurements 0.5 mg of rice leaves collected from the hydroculture experiment were freeze-dried,

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ground and sifted through a 100-mesh sieve for Fourier Transform Infrared (FTIR)

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measurements. The Bruker Vertex 70 (Bruker) spectrometer using attenuated total

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reflection (ATR) technique was used to obtain the FTIR spectrum. The FTIR spectra from 4000-400 cm-1 were acquired in transmission geometry by 64 scans at 4 cm-1

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spectral resolution (Depciuch et al., 2017).

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2.4 TEM measurements

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A rectangular fragment (1 mm × 2 mm) was cut from the flag leaf tips from the hydroculture experiment, and the cutting position was selected to include the vein. The fragment was immediately fixed in 0.2 M PBS (pH 7.2) with 4% (v/v) glutaraldehyde. The leaf samples were then rinsed in 0.1 M PBS, then fixed in 1% OsO4 in PBS, and rinsed again with buffer. They were then dehydrated in fractionated acetone series and buried in epon 812. The polymerization was conducted at 30 °C, 24 hours; 40 °C, 24 hours; 60 °C, 48 hours respectively. Ultrathin slices were made with LKB-8800 Ultramicrotome, stained with uranyl acetate and lead citrate, and examined under a Quanta 200 transmission electron microscope (TEM) (Netherlands). Each 9

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2.5 Data analysis Statistical analysis was calculated using SPSS Statistics (version 19, IBM Corp., Armonk, NY, USA). Treatments of S at different rice growing stage were tested by one-way analysis of variance (ANOVA). The effects of foliar treatment and blank treatment at different Cd concentrations were tested by multi-factor ANOVA. The

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treatment effects at different Cd concentrations were compared by using the least

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significant different test with the p-value less than 0.05. The effects with foliar

3. Results and discussion

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Origin.

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treatment and blank treatment were tested by T - test. Plots were generated using

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The effects of foliar application of S on rice under Cd stress were studied by

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two-season field experiments. After the positive effect of S was observed on

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decreasing the concentration of Cd in grain and promoting the rice growth, the hydroculture experiment was conducted to clarify the possible mechanisms related to S-induced Cd tolerance in rice.

3.1 Effect of foliar application of S on grain Cd and grain yield In early rice and late rice field experiments, foliar application significantly increased grain yield as compared with the control (Figure 1A and 1C). The highest increase of grain yield was 17% with foliar application at tillering stage for early rice, and 30% with foliar application at flowering stage for late rice. For early rice, foliar application significantly decreased grain Cd as compared with the control. The 10

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decrease trend of grain Cd was consistent with the increase trend of grain yield for all different treatments. The highest decrease rate of grain Cd occurred at the tillering stage as 41% (Figure 1B). For late rice, the grain Cd significantly decreased by 27% and 48% with foliar application at flowering and tillering stage respectively (Figure 1D).

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The low rice production in controls may be due to the increased Cd concentration

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in rice. Adverse effects on rice ultra-structures from Cd disturbed the normal function

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of rice and inhibited rice growth. The positive role of foliar application on plant

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growth has been proved in agricultural practices (Tatagiba et al., 2016). It was

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suggested that foliar application of biostimulants was beneficial to improve plant vitality, and then improve plant resistance against diseases and stress, as well as grain

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yield (Teixeira et al., 2017). Foliar application of mineral elements is also a

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bioaugmentation technology recommended for increasing yield. First, it increases the

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production of healthy substances, such as ascorbate, cysteine-rich polypeptides, amino acids, which increase the absorption of essential mineral elements and change the uptake of heavy metals by plants. Second, it reduces the generation of antinutrients, such as oxalate, polyphenolics, which also affect the uptake of heavy metals (White and Broadley, 2009). Foliar application of S may act as a source of nutrition for plants, thus increasing the length of tillers, TKW, plant height, nutrition, and yield (Na and Salt, 2011). The increased plant growth may come from the decreased oxidative stress and direct damage to rice by the low Cd concentration after foliar dressing. The concentration of 0.5 g L-1 for foliar application of S was suitable for the crop quality 11

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and productivity (Roosta and Hamidpour, 2011; Araujo et al., 2017; Seleiman and Kheir, 2018). The effective results at tillering stage were probably due to the fact the most efficient uptake of nutrients by rice at the tillering stage (Ma and Takahashi, 1990). Foliar application at the tillering stage was the best time to alleviate the toxicity of Cd, which was in agreement with the report of Liu et al (2009). They found

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that Si applied at tillering stage had highest efficiency in reducing the toxicity of Cd

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in rice.

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3.2 Effect of foliar application of S on the photosynthesis system

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and chlorophyll fluorescence

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Figure 2 displays the photosynthetic parameters of Pn, Gs, Ci and Tr of rice leaves under different concentrations of Cd. The values of Pn, Gs, Ci and Tr significantly

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decreased with the increase of Cd concentration. The values of Pn, Gs, Ci and Tr with

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foliar treatments increased by 2 - 25%, 7 - 25%, 2 - 10% and 1 - 11% as compared

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with those blank foliar treatments (with distilled water). The highest increase rate was found in the rice treated with 0.2, 0.5, 1.0 mg L -1 Cd. As the concentration of Cd further increased, photosynthesis parameters reached the saturation point. The

beneficial

effect

of

foliar

application

on

plants

was

obviously

concentration-dependent (Zong et al., 2017). The foliar dressing of S was effective to maintain high photosynthetic characteristics under low or medium Cd contamination. Cd may affect plant growth by changing chlorophyll synthesis, transpiration process, respiration process and stomatal opening, thus reducing photosynthesis (Farquhar and Sharkey, 1982). Stomatal closure was one of the earliest responses to Cd stress and 12

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decreased photosynthesis (Ashraf and Harris, 2013). Gas exchange parameters (Ci and Tr) are the limiting factors in the diffusion and immobilization of CO2, which are related to the activity of CO2 immobilized enzyme, ribulose bisphosphate carboxylase and oxygenase (RuBisCO) (Hou et al., 2018). The toxicity of Cd can be mediated by increasing the carboxylation efficiency of RuBioCO (Nwugo and Huerta, 2010). It is

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reported that Pn is a determinant of plant growth and biomass production (Lu et al.,

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2015). Christy and Porter (1982) showed a positive correlation between Pn and yield

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through repeated multi-quarter monitoring experiments. The results of Cao et al (2015)

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indicated that the value of Pn can be used to estimate grain yield. In this study, the

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increased Pn value after foliar treatments may be attributed to the increase of Gs, Ci and Tr, thereby accelerating the effective carbon assimilation period of rice leaves and

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thus accelerating the accumulation of photosynthetic products.

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Figure 3 displays the chlorophyll fluorescence parameters of rice leaves under

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different concentration of Cd. Cd stress significantly diseased the values of Fv/Fm, PSII and qP in rice, while NPQ value increased. When compared with the control, the application of S increased Fv/Fm, PSII, qP values by 3 - 9%, 3 - 21%, 3 - 9%, and decreased the NPQ value by 2 - 17%. However, as the concentration of Cd increased to more than 1.0 mg kg-1, the foliar treatment tended to show no effect. Although the effect of S on chlorophyll fluorescence in rice leaves has not been reported, it has been confirmed that the changes of chlorophyll fluorescence can reflect biological or abiotic stresses (Mehta et al., 2010). The decrease of Fv/Fm, PSII and qP indicated that the photo-activation of PSII was inhibited by the toxicity 13

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of Cd, which was resulted from the destruction of antennae pigments, the restriction of electron transport from PSII to PSI, and the destruction of the structural integrity of thylakoid membrane. On the contrary, the decrease of NPQ suggested the increasing of photochemical reaction efficiency, and more absorbed light energy can be used for photochemical reaction and for the synthesis of energy or carbohydrate (Li et al.,

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2014). In this experiment, the foliar application of S increased the quantum yield and

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protected the photosynthetic system from damage. These findings were also

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consistent with the results of photosynthetic parameters in rice.

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3.3 FTIR characterization for the effect of foliar application of S

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at different concentrations of Cd for rice FTIR has been used as a successful tool in monitoring structural changes of organic

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molecules (Gul et al., 2016; Wang et al., 2018). Fig. 4 showed the FTIR spectra of

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rice leaves with and without foliar application of S. The characteristic absorption

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bands corresponding to functional groups were listed in Table 1. The absorption of C-O, O-H, C=C, C-H, N-H all decreased with the increased concentration of Cd when compared with the control. After foliar treatments, the absorption increased for the above mentioned five chemical bonds, indicating the more stimulated production of polysaccharides (1032-1041 cm-1; 1238-1386 cm-1), lignin and carboxylate (1600-1630 cm-1), lipids and aliphatic acid (2850-2924 cm-1), protein and nucleic acid (3336-3386 cm-1). Lignin, lipids and aliphatic acid are the main components of cell wall and cell membrane, and are considered as the key barrier to protect protoplast from Cd 14

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poisoning (Zong et al., 2017). The foliar application of S also stimulated the synthesis of polysaccharides, carboxylate and several proteins in leaves, which might provide a number of potential ligands to sequestrate Cd in organelles, such as vacuoles. Previous studies reported that the synthesis of phenolic acids in wheat leaves was stimulated by foliar treatment (Reddy et al., 1999). Rizwan et al (2017) also found

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that the reduction of Cd concentration with foliar application of aspartic acid may be

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due to the excessive production of phenolic compounds in rice seedlings, thereby

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reducing the uptake of Cd in plants. These phenomena indicated that the pollution of

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Cd disturbed the normal function of rice, and inhibited the growth of rice. Foliar

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application enhanced synthesize of organic molecules, increased rice resistance, rice quality, and yield.

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mesophyll cells

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3.4 Effect of foliar application of S on the ultrastructure of

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The TEM micrographs showed the ultrastructural alterations of rice leaves (Fig.5). The cell shape in the control group was normal, the cell wall was smooth and continuous, the nuclei and nucleoli were large, and the chloroplasts were abundant. However, cells treated with Cd displayed series of morphological changes, such as contraction, deformation and rupture. Some cellular organs disappeared, which might be important organelles for the synthesis and transport of protein and lipid molecules (Ren et al., 2014). After Cd treatment, Cd began to display as electron-dense particles (Zheng et al., 2012; Mitra et al., 2018). With the increase of Cd concentration, more and more irregularly thickened electron-dense sediments appeared. When treated with 15

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foliar application, cell walls and vacuoles were the most common electron-dense deposits in mesophyll cells. With the increase of Cd concentration above 1.0 mg kg-1, the sediments randomly scattered in the whole cell, indicating the foliar treatment could not deal with the high level of Cd. Cd exists in both forms of free Cd2+ and wall-bound deposition. The chemically

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heterogeneous walls by Si modification could provide more binding sites for the Cd 2+.

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The mechanism of co-deposition of Si and Cd in the cell walls explained the

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inhibition of Cd2+ uptake in rice (Liu et al., 2013). The effect of S addition was also

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confirmed in Hg-contaminated paddy soil by decreasing Hg motility via the

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conversion of RS-Hg-SR to HgS (Li et al., 2017). In this study, Cd was mainly immobilized in vacuoles and cell walls, binding to the induced proteins when the

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leaves were sprayed with S (as described in Section 3.3). Sulfur is an essential

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component of key amino acids for the synthesis of proteins (Duncan et al., 2018).

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Sulfur-containing compounds (such as GSH, PCs) are crucial in detoxifying heavy metal stress. Foliar application of S mediated the suppression of Cd-induced oxidative stress, via Cd sequestration into compartments including cell walls and vacuoles.

4. Conclusion This study provided preliminary evidence that the effect of foliar application of S in Cd-stressed rice might be related to the regulation of photosynthesis and protein synthesis. First, the foliar spray of S alleviated Cd toxicity in PSII reaction center and increased the photosynthetic efficiency. Second, it stimulated the synthesis of polysaccharides, carboxylate and several proteins in leaves, which provided a number 16

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of potential ligands to sequestrate Cd in organelles, mainly cell walls and vacuoles, and then blocked the path of Cd into the structure in plants. Sulfur likely retained the structure of plant cells and effectively regulated protein expression by alleviating Cd stress without sacrificing yield. As the concentration of Cd reached to higher levels, the foliar treatment inclined to have no effect, indicating that the foliar treatment was

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not suitable for the high Cd levels. Therefore, two measurements could be taken to

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optimize this technology. (1) Using surfactant to increase the solution penetration and

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spreading for the best foliar dressing efficiency; (2) Using the combination treatments

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to alleviate the limited effect of any single treatment. For example, stopping the use of

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polluted irrigation water could be combined with the foliar treatment to reduce the grain Cd, soil available Cd, and also increase the rice yield. Further studies may also

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explore the effects of leaf application of S on stress tolerance and gene / protein

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expression in different rice organs.

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Acknowledgements

This work was supported by the National Key Research and Development Program of China (2017YFD0801302).

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Conflict of interest statement The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work

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submitted

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(B)

c

Early rice

a

Yield (kg ha-1)

0.6

b

a

d

6000

0.4

c

3000

0.2

0

l

o ntr Co

e tag

s ling ed Se

(C)

ge

ta gs rin

le Til

l

o ntr Co

e tag

we Flo

c

b

c

(D)

e tag

s ling ed e S

gs rin

e tag

le Til

gs rin we

e tag

0.0

Flo

Late rice

a

0.6

f

9000

gs rin

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a

c

pr ol ntr Co

lin ed Se

ta gs

e-

3000

0

0.4

b

ge

ge ge sta sta ing ng i r r e le w Til Flo

l

o ntr Co

s ling ed Se

0.2

e tag

ge

le Til

ta gs rin

e tag

0.0

gs rin

we Flo

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Yield (kg ha-1)

a

6000

Grain Cd (mg kg-1)

c b

Grain Cd (mg kg-1)

(A) 9000

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Fig. 1 Effect of foliar application of 0.5 g L-1 S at different rice growing stages on the

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grain yield and grain Cd concentration of early rice (A, B) and late rice (C, D) in field

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trial. The bar represents the standard error of three replicates. The letter on the top of bar indicates the significant difference at P < 0.05.

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Aa

(B)

Bb

Bb

Bc

Aa

-S +S

Bd

12

Aa

0.3

Bc Ab

Ab

0.2

Ad

Ac

8

Ad

Ae

Ac

Ae

Af Ae

4

Ad

Af

Ae

0.1

Ae Af

0

0.0

Bb Bb

Bb

Aa Aa Bc

280

Ae

Ac Ad Ae

240

(D)

4.0

Bb

Bc

Ab

Ad

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Ab

e-

Ci (mol CO2 mol-1)

Aa

(C)

360

f

Aa

Control Cd/0.2 Cd/0.5 Cd/1.0 Cd/2.0 Cd/5.0

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Control Cd/0.2 Cd/0.5 Cd/1.0 Cd/2.0 Cd/5.0

320

Gs (mol H2O m-2 s-1)

(A)

Aa

Af

3.0 Ac

Ad Ae Ad Ae

2.0

Control Cd/0.2 Cd/0.5 Cd/1.0 Cd/2.0 Cd/5.0

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Control Cd/0.2 Cd/0.5 Cd/1.0 Cd/2.0 Cd/5.0

Ab

Tr (mmol H2O m-2 s-1)

Pn (mol CO2 m-2 s-1)

16

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Fig. 2 Effect of foliar application of S (0.5 g L-1 dose supplied at tillering stage) at

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different concentrations of Cd on the net photosynthetic rate (Pn) (A), stomatal

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conductance (Gs) (B), intercellular CO2 concentration (Ci) (C), and transpiration rate (Tr) (D) in rice. The bars indicate the standard error of the mean. Capital letters indicate significant differences at p < 0.05 between foliar treatment and non-foliar treatment. Small letters with blue color indicate significant differences at p < 0.05 between different concentrations of Cd treatment under foliar application of S. Small letters with green color indicate significant differences at p < 0.05 between different concentrations of Cd treatment with no foliar application.

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Aa

Aa

(A)

Bb Bb

Aa

0.4

Bb

0.75

Fv/Fm

(B)

Bc

Bc

Aa

0.70

0.3

Bd

Ab

Bd

Ab Ae

Ac

Ac Be

0.65

-S +S

PSII

0.80

0.2

Ad

Af Ad Ae

Ae Af

Ae

0.1

Control Cd/0.2 Cd/0.5 Cd/1.0 Cd/2.0 Cd/5.0

Control Cd/0.2 Cd/0.5 Cd/1.0 Cd/2.0 Cd/5.0 Af

(C)

Bb

Ae

(D)

Aa

qP

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Bc

Ac

Bb

Ad Ae

Ad

Aa

Ae

0.3

Af

Ae

0.4 Ad

Ac

Ab

0.2

e-

Aa

0.6

Bd

Bc

pr

Ab

0.4

Af

f

Bb

0.5

NPQ

Aa

Control Cd/0.2 Cd/0.5 Cd/1.0 Cd/2.0 Cd/5.0

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Control Cd/0.2 Cd/0.5 Cd/1.0 Cd/2.0 Cd/5.0

Fig. 3 Effect of foliar application of S (0.5 g L-1 dose supplied at tillering stage) at

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different concentrations of Cd on the maximum photochemical efficiency of PSII:

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Fv/Fm (A), actual PSII photochemical efficiency (PSII) (B), photochemical

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quenching coefficient (qP) (C), and non-photochemical quenching coefficient (NPQ) (D) in rice. The bars indicate the standard error of the mean. Capital letters indicate significant differences at p < 0.05 between foliar treatment and non-foliar treatment. Small letters with blue color indicate significant differences at p < 0.05 between different concentrations of Cd treatment under foliar application of S. Small letters with green color indicate significant differences at p < 0.05 between different concentrations of Cd treatment with no foliar application.

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3200

2400

1600

800

T%

0.999

E

Control Cd/5.0+S Cd/5.0

D

Control Cd/2.0+S Cd/2.0

0.972 0.945

T%

0.999 0.972 0.945

0.945

C

f

Control Cd/1.0+S Cd/1.0

0.972

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T%

0.999

0.930

B

Pr

Control Cd/0.2+S Cd/0.2

0.961

4000

A 3200

2400

1600

al

T%

0.992

0.930

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Control Cd/0.5+S Cd/0.5

0.961

e-

T%

0.992

800

-1

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Wavenumber (cm )

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Fig. 4 FTIR spectra of rice leaves treated with foliar application of S (0.5 g L-1 dose supplied at tillering stage) at different concentration of Cd in hydroponic medium

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Journal Pre-proof Cd/0.2

Cd/0.5

Cd/1.0

Cd/2.0

Cd/5.0

Cd/0.2+S

Cd/0.5+S

Cd/1.0+S

Cd/2.0+S

Cd/5.0+S

e-

pr

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f

Control

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Fig. 5 TEM images of rice leaves treated with foliar application of S (0.5 g L-1 dose

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supplied at tillering stage) at different concentrations of Cd in hydroponic medium

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Table 1 Assignment and characterization of measured absorption bands (Basnet et al., 2016; Depciuch et al., 2017; Zhang et al., 2019) Wavenumb Functional group vibration

Possible origin

1032-1041

C-O and O-H group stretching

Polysaccharides

1238-1386

C-O group stretching

Polysaccharides

-1

f

er (cm )

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Aromatic C＝C stretching (conjugated), 1600-1630

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Lipids, aliphatic bonds Polysaccharides,

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Pr

O-H and N-H group stretching

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3336-3386

Antisymmetric C-H stretching (CH3 groups)

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2850-2924

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Asymmetric C-O stretch in COO

Lignin or carboxylate

-

31

protein, nucleic acid

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Highlights

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(1) Foliar treatment of S was effective for reducing Cd in rice and increasing the grain yield; (2) Foliar spray of S alleviated Cd toxicity on PSII reaction center and increased photosynthetic efficiency; (3) Foliar spray of S stimulated the syntheses of organic molecules which potentially sequestrate Cd in vacuoles and cell walls.

32

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5