Salicylic acid application alleviates Cadmium accumulation in brown rice by modulating its shoot to grain translocation in rice

Cadmium (Cd) contamination has been recognized as a major threat to the agricultural system and crop production which posing serious threat to human health. Salicylic acid (SA) serves as an important signaling molecule and plays an important role in against Cd toxicity. In the previous field experiments, we found SA spraying could reduce the Cd accumulation of rice grain grown in Cd-contaminated soil. This study investigated the effects and mechanisms of SA spraying on leaves of rice seedlings under Cd stress. Results showed that SA treatment could alleviate the Cd toxicity of rice not by changing the physical and chemical properties of the soil, but by increasing the activities of antioxidant enzymes to reduce the H 2 O 2 accumulation in rice. And the key factor of SA treatment reducing Cd accumulation in rice grain was the decreasing of Cd contents in rice leaves at the flowering stage. This indicated that SA could modulate the Cd accumulation of shoots to reduce the Cd translocation to rice grain. Furthermore, SA could increase the H 2 O 2 contents in a short-term to activate the SA-signaling pathway, and modulate the expression levels of Cd transporters ( OsLCT1 and OsLCD ) in rice leaves toraise Cd tolerance and reduce Cd accumulation in rice grain. Thus, SA spraying can be used as an effective measure to cope with Cd contamination in paddy soils.


Introduction
Growing population and fast industrialization coincide together, results in the generation and dissemination of huge amount of toxic metals in the environment (Hasanuzzaman et al., 2012). Cd is one of the most phytotoxic heavy metals which have no biological role in plants, and its contamination in agricultural soil has become a great problem for crop production in recent decades. As a common soil pollutant, Cd can accumulate in plant tissues to cause toxicity and enter human through the food chain and drinking water to cause diseases in kidney, bone and respiratory system and cancers (Zhou et al., 2017). Rice is an important staple food for nearly half of the world's population (Valipour et al. 2015) and is also a major source of Cd uptake for people. Consumption of Cd-contaminated rice caused the outbreak of "Itai-Itai disease" in the mid-20 th century in Japan. Recently, Cdcontaminated rice has also occurred in China, which poses important public health risks (Liu et al., 2016b). Therefore, it is essential to produce low-Cd rice to reduce the potential risks that Cd poses to human health. In the previous field experiments, we found that SA spraying could reduce the Cd accumulation of rice grain grown in Cd-contaminated soil. However, the study on the reduction of Cd accumulation in rice grain by SA application is still blank. Thus, we conducted a pot experiment in the laboratory to screen the best concentration of SA treatment, and declare its molecular and physiological mechanism in regulating Cd accumulation of rice.

Growth Conditions and Experimental Design
Seeds of the pre-screened as a high grain-Cd-accumulating rice (Xiushui 110) was used in the pot experiment. After germination, rice seedlings were grown in a grid until to 4-leaf stage (Wang et al., 2009), then the plants were transplanted into the plastic pots filled with 10.0kg soil, four seedlings were grown in each pot. After treated with Cd for 1 week, the pre-screened 0.1mM SA solutions and 0.1mM AOPP (L-2-aminooxygen-3-phenyl acrylic acid, synthetic inhibitors of SA) were sprayed on the seedlings' leaves until the whole leaves were covered by the solutions at the tillering stage, heading stage and flowering stage, respectively. The experimental groups were set up 6 experimental groups: CK (control, without Cd or SA treatment), Cd (single Cd treatment), SA (single SA treatment), SA+Cd (treated with SA under Cd stress), AOPP (treated with AOPP), AOPP+Cd (treated with AOPP under Cd stress).

Estimation of the agronomic traits of rice
Samples were collected from each pot at the growth stages after sprayed with SA (or AOPP) for 5 days and at the maturity stage, respectively. The rice plants height and root length were measured by a ruler. The dry matter accumulation was determined as follows: The plants were divided into roots and shoots, then dried in the oven for 2h at 105℃ and baked at 70℃ to constant weight. The 1000-grain weight was also analyzed.

Estimation of the pH, available Cd and total Cd in soil
The soils at different stages of rice plants were used in the determination, and the sampled soil was naturally air-dried and removed through a 2.5 mm nylon screen to remove grit and plant debris, then passed through a 100-mesh screen and stored.
The pH, available Cd and total Cd were determined according to Wang et al. (2018).

Estimation of Cd contents in different parts of rice
Different parts of rice samples were taken at heading and flowering stages after sprayed with SA for 5 days and at the maturity stage, respectively. Roots of rice samples were immersed in 20mM disodium ethylenediamine tetra-acetic acid (Na 2 -EDTA) for 20min, and then rinsed three times with deionized water. All the rice samples were dried at 105℃ for 2h firstly , then baked at 70℃ to achieve a constant weight, and finally ground to powders. Each 0.3g dried rice powders were treated with 6mL HNO 3 (brown rice) or 6mL HNO 3 plus 0.5ml H 2 O 2 (other rice samples) to digest in a microwave digester (CEM-MARS, Boston, USA). After digestion, all the mixtures were driven acid to 1mL at elevated temperature. The solutions were then diluted into 25mL volumetric flasks with ultra-pure water, and the clarified samples thus obtained were kept in a refrigerator at 4 ºC for further analysis. The Cd contents were determined by an atomic absorption spectrometer (AA7000, SHIMADZU, and Kyoto, Japan).

Estimation of endogenous SA content and PAL activity
Fresh leaves (0.1g) were ground with liquid nitrogen into power, and added 0.5mL sterile water, then carried out ultrasonic 30min. The homogenate was placed in centrifuge at 10,000rpm for 10min, and the supernatant obtained was the sample extract. Finally, the endogenous SA content was measured by the Plant Salicylic acid (SA) ELISA Kit (JM-E100150, U.S.A TSZ biological Trade Co., Ltd). The determination of PAL activity was followed with the instructions of the specific Kit (A173, Nanjing Institute of Bioengineering).

Gene expression analysis using qRT-PCR
Total RNA isolated from the top 2nd leaves of rice plants after SA sprayed for 5days at the flowering stage, and the different cDNA were synthesized with the primerscript M RT reagent kit (Takara, Japan). Primer sequences used were listed in Table 1, β-actin was chosen as the internal control. The qRT-PCR was performed using a Rotor-Gene Q machine (parameters: 10min at 95℃, followed by 45 cycles of 10S at 95℃, 15S at 58℃, and 15S at 72℃). All the reactions were performed in triplicate (technical replicates) for each biological replicate (three for each treatment). Quantification of gene expression was calculated using △△ CT method (Wang et al., 2012).

Statistical analysis
The experimental data was analyzed using the Prism software (Graphpad Prism 6.0, GraphPas Software Inc., SanDiego, California, USA) and presented as the mean ± standard deviation (SD) which calculated with at least three replicates. Student's ttest was adopted for the analysis of significant differences among the treatments. P-value less than 0.05 were considered as significant.

SA application enhances the tolerance to Cd in rice
Preliminary experiments showed that SA played an important role at the reproductive growth period (data not shown). Therefore, our experiments were conducted mainly at the reproductive growth stages (heading stage, flowering stage and mature stage). Cd stress obviously reduced seedlings growth, which mainly reflected in the following aspects: root length, plant height, shoot Dw plant-1, and1000-grain weight. SA application could relieve the Cd toxicity obviously, increased the plant height and shoot Dw plant-1 evidently of the Cd-exposed rice plants, but there was no significant difference between Cd and AOPP+Cd groups (Table 2 and 3), the same trends were observed at all the three stages of rice plants. In addition, application of SA or AOPP to non-stressed seedlings did not result in any difference in agronomic traits.

SA application changed the physical and chemical properties the soil
From the analysis of the effects of SA and AOPP application on the total Cd content, available Cd content and pH value of the soil, we found the total Cd and the available Cd contents of the soil were decreased continuously during the growth of rice. And the exogenous SA or AOPP treatment did not affect the total Cd and the available Cd contents at the four different stages obviously except there was a significant difference between SA+Cd group and AOPP+Cd group at the mature stage. Furthermore, the pH value of the Soil also was not changed after the treatment with exogenous SA or AOPP (data not shown).

SA application induces low Cd accumulation in rice grain
Cd accumulation of rice grain (0.29mg/kg) in Cd-exposed rice plants was significantly greater than the regulatory Cd limit (0.2mg/kg), but SA application could reduced the accumulation of Cd to 0.12mg/kg. Meanwhile, the AOPP+Cd group increased Cd accumulation (0.34mg/kg) significantly which was higher than that of the Cd group ( Figure 1).

SA application decreases the Cd contents in other parts of rice
The rice plants accumulated significant amount of Cd in different parts during Cd stress, and the roots accumulated the most Cd by nearly 80%. Compared with the Cd-exposed rice plants, application of SA could reduce the Cd accumulation of leaves and flag leaves remarkable, especially the leaves at flowering stage (around 45% reduction), resulted in around 53% reduction of Cd accumulation in panicle.
However, SA application had no significant impact on Cd accumulation in roots, stems and nodes. Furthermore, AOPP application significantly enhanced the total Cd accumulation in panicle, and the main reason was that the Cd contents in leaves at flowering stage were increased ( Figure 2). Therefore, we indicated the main reason of SA treatment could effectively reduce Cd content in brown rice and panicles was SA could significantly reduce Cd contents in rice leaves at flowering stage. Different treatments could affect the mobility of Cd in rice. From the analysis of effect of the three groups (Cd, SA+Cd and AOPP+Cd) on the Cd mobility in rice, we only found the Cd mobility at flowering stage (stem-to-leaf and leaf-to-panicle) and mature stage (leaf-to-panicle) were shown obvious significance (Table 4). SA application could markedly reduce the Cd mobility, as well as AOPP application could markedly increase the Cd mobility in stem-to-leaf at flowering stage.
Furthermore, the different Cd mobility resulted in the different Cd accumulation in rice panicles, SA application both reduced the Cd mobility in leaf-to-panicle approximately both 41% at flowering stage and mature stage, and AOPP application increased the Cd mobility in leaf-to-panicle about 47% at flowering stage and 29% at mature stage separately. Therefore, leaves at flowering stage were the critical parts for SA regulate Cd accumulation in rice grain.  (Figure 4). The expression of OsPAL in SA group were higher than that of CK group, it could arrived at more than 2 times at 6 hours after SA application at the flowering stage, as well as the expression level of OsPAL in SA+Cd group were more than 2.5 times than that of Cd group at 6 hours. In contrast to the changes of OsPAL expression level, there were no significance of the expression levels of OsICS between CK and Cd group, but the expression levels of OsICS in SA (or SA+Cd) group could increase by more than 4 times than that of WT (or Cd) group, respectively. OsWRKY7 and OsWRKY70 were negatively correlated with the SA contents in rice.

SA application sustains a high level of H 2 O 2 and boostsantioxidant enzymes' activities under Cd stress
In order to examine whether SA induces H 2 O 2 production in rice leaves during Cd stress, the contents of H 2 O 2 and enzyme activities of CAT, POD, and APX in the rice leaves at the flowering stage were determined. Results showed that Cd stress could result in a high H 2 O 2 accumulation. The H 2 O 2 content in SA group was reduced in a short period of time after SA application, the H 2 O 2 content was lowest on the 1st day, then increased continuously, and returned to the same level as the control on the 5th day, similar trend was observed in SA+Cd group. Furthermore, the H 2 O 2 content in SA+Cd group was higher than that of the Cd group temporary, but after SA application it decreased with time to the lowest level on the 3rd day, and then increased to the level similar with the Cd group on the 5th day ( Figure 6).
Furthermore, compared with the Cd group, the activities of antioxidant enzymes (such as SOD, POD and CAT) in SA group and Cd+SA group showed a similar enhancement moderated by SA treatment (Figure 6). It was obvious that SA application on rice leaves during Cd stress could increase the H 2 O 2 content in a short-term and improve the Cd tolerance of rice.

Conclusion
In conclusion, it is evident from the present work that SA can effectively boost the antioxidant enzymes activities and SA signaling pathway to response to Cd stress in rice. SA spraying modulates the Cd mobility of stem-to-leaf and leaf-to-panicle in rice at flowering stage to reduce the Cd accumulation in rice grain which is helpful in developing the low-Cd rice grain (Figure 8).

Declarations
Ethics approval and consent to participate: No applicable.

Consent for publication: No applicable.
Availability of data and material: All essential data are part of the article.    Tables   Table 1 qRT-PCR primers for Gene expression analysis in leaves of rice plants Primer names Sequence (5'-3')  Effects of different treatments on Cd accumulation in brown rice at mature stage Effects of SA treatment on the expression levels of OsPAL and OsICS in rice leaves at floweri Effects of SA treatment on the expression levels of OsNPR1, OsWRKY45, OsWRKY7, and OsWR  Effects of SA treatment on the expression levels of OsLCT1and OsLCD in rice leaves at flower