Root Aeration Promotes Cadmium Accumulation in Rice by Regulating Iron Uptake-Associated System

Huang Qina, Wu Yinliang, Shao Guosheng

Research Paper

Root Aeration Promotes Cadmium Accumulation in Rice by Regulating Iron Uptake-Associated System

Huang Qina1, Wu Yinliang2, Shao Guosheng1

(China National Rice Research Institute, Hangzhou 310006, China; Ningbo Academy of Agricultural Sciences, Ningbo 315040, China)

Different cadmium (Cd)-accumulated rice genotypes (Erjiunan 1 and Fupin 36) were used to explore the effect of rice rhizosphere aeration on Cd uptake and accumulation. Aeration in the nutrient solution influenced the agronomic characteristics induced by Cd-stress, such as the increases of rice root length and root vigor, but the reductions of plant height and shoot dry weight. Aeration also alleviated the decreasing effects of Cd stress on antioxidant enzyme activities and soluble protein, malonaldehyde and nicotianamine contents in rice. Moreover, with aeration treatment, the accumulation and bioavailability of metal elements changed significantly, with a Cd increase and an Fe reduction in both rice genotypes. In addition, at the molecular level, aeration upregulated the expression of Fe-inducible genes (such as,,and). Furthermore, as a Cd2+/Fe2+transporter, the high transcription level ofcan elevate the Cd uptake and translocation in rice due to the Fe reduction caused by aeration and Cd-exposure, which indicated thatmight play a crucial role in the effect of aeration on Cd uptake and accumulation.

aeration; antioxidant enzyme; cadmium; gene regulatory network; rice

As a nonessential element in plant development, cadmium (Cd) is not necessary for any physiological functions. It inhibits the growth and development of plants and causes serious symptoms, including chlorosis, growth retardation, root tip browning and even death under high-Cd level stress (DalCorso et al, 2008; Clemens and Ma, 2016).Cd exposure in plants can destroy the redox homeostasis system and induce an imbalance in the activities of antioxidant enzymes, accelerate the accumulation of reactive oxygen species (ROS), and trigger the H2O2and O2·?overaccumulation (Romero-Puertas et al, 2004). In addition, Cd stress also damages oxygen utilization in the electron transportchain, disrupts the cell redox and ion transport balance, and causes phytotoxicity in plants (Shahid et al, 2017). Similarly, Cd has a strong harmful effect on the human body through the food chain. Rice, as one of the main food crops in Asia, exhibits a relatively easy absorption and accumulation of Cd. Cereal grains are the dominant source of Cd in the human diet and are considered as the main Cd source for Asian people (Meharg et al, 2013).Therefore, the control of Cd pollution in rice has become a major issue worldwide.

In the process of rice production, water management is considered as an important measure for controlling Cd pollution. Flooding can significantly decrease Cd accumulation by reducing the phytoavailable Cd content and/or root Cd uptake capacity. However, intermittent (via the release of ponded water) and moist irrigation cause soil oxygenation (increasing the content of soluble Cd and converting soluble iron (Fe)into insoluble Fe form) and significantly enhance the absorption and translocation of Cd (Huang et al, 2013). In fact, changes in oxygen concentration lead to changes in the redox state of rhizospheres or soil, which is a key factor in water management for controlling Cd pollution.Aeration can significantly affect the Cd uptake in plants.For instance, aeration promotes rice seedling growth, delays root senescence, and increases Cd retention in roots to alleviate Cd toxicity (Li et al, 2019). Huang et al (2019) found that aeration changes the morphology and anatomy of rice roots and promotes Cd absorption. The root morphology of maize affects Cd accumulation depending on the cultivation conditions, such as hydroponic, aeroponic and soil-based growth (Redjala et al, 2011). However, aeration pretreatment enhances the secretion of oxygen in root tips and improves the tolerance ofseedlings to Cd stress (Xin et al, 2019). Moreover, the oxygen in rhizosphere oxidizes Fe2+to Fe3+(iron hydroxide) and forms iron plaques. It has been widely reported that Fe plaques can immobilize Cd, lead (Pb) and nickel (Ni) on root surfaces and effectively reduce their translocation from roots to shoots (Tripathi et al, 2014). However, the underlying substantive mechanisms between aeration and Cd accumulation in plants have not been explored, especially those associated with the effect of aeration on the Cd uptake/transport capacity of plant roots.

Rice roots mainly uptake Cd via divalent metal ion transporters, such as Fe2+, Mn2+, Zn2+and Ca2+, due to the weak selectivity of these transporters for substrates (Yoneyama et al, 2015). To date, a large number of divalent metal ion transporter genes with Cd absorptioncapacity have been cloned in rice. The natural resistance- associated macrophage protein (NRAMP) family are responsible for divalent metal ions (Fe2+, Mn2+and Cd2+, etc.) transport from the environment and/or among plant tissues. In, OsNRAMP1, which belongs to the NRAMP protein family andlocalizes to the plasma membrane, participates in the cellular uptake of Cd2+and Fe2+, and is significantly upregulated by Fe deficiency (Takahashi et al, 2011). Another NRAMP family protein, OsNRAMP5, is constitutively expressed in rice plants and is the predominant pathway for Mn2+and Cd2+uptake with a small contribution to Fe2+uptake (Ishimaru et al, 2012). Therefore, the dysfunction ofcan severely perturb Mn homeostasis inplants (Ishimaru et al, 2012). The rice heavy-metal P-type ATPase family (OsHMA2 and OsHMA3) are responsible for Cd distribution in plant tissues (Satoh- Nagasawa et al, 2013). Similar to the Fe2+uptake systems in dicotyledons, Fe uptake transporters (OsIRT1 and OsIRT2) are involved in the influx of Cd2+and Fe2+(Ishimaru et al, 2006). Moreover,has been revealed to play a role in Cd transport from the phloem to grains (Uraguchi et al, 2011). In addition, other genes related to Cd tolerance have been reported in rice, such as,,and(Kuramata et al, 2009; Ishikawa et al, 2010; Shimo et al, 2011; Song et al, 2015; Liu et al, 2019).

In this study, using two rice genotypes (Erjiunan 1, EJN1 and Fupin 36, FP36) with differences in Cd- accumulated grains explored the effects of the molecular mechanisms on the oxygen promotion of Cd uptake and accumulation in plants. The agronomic characteristics,antioxidant enzyme activities, malondialdehyde (MDA) content, soluble protein content and the transcript levels of metal ion-transport related genes in both rice genotypes were assessed under hydroponic conditions. Based on these results, our study might provide the fundamental information to explain the higher Cd accumulation in rice under intermittent/moist irrigation than under flooding cultivation.

RESULTS

Effects of Cd and aeration on rice agronomic traits

The agronomic traits (including plant height, root length and dry weight) were significantly affected by the Cd and aeration treatments (Table 1, Table S1 and Fig. S1). Under 1.0 μmol/L Cd stress, plant growth was seriously inhibited, and plant height, root length, shoot dry weight, root dry weight and root vigor were reduced at 10 and 20 d under nonaeration and aeration conditions (Table 1 and Table S1). However, there were differences in plant growth parameters between 0 and 1.0 μmol/L Cd under aeration conditions. Under aeration conditions, it was resulted in the increases of rice plant height, root length, root vigor and dry weight (roots and shoots) in EJN1with non-Cd stress. Nevertheless, compared with nonaeration, under 1.0 μmol/L Cd stress, plant height and shoot dry weight were significantly reduced under aeration conditions, indicating that aeration can strengthen the effect of Cd toxicity on rice growth (Table 1). Furthermore, Cd stress can significantly inhibit root vigor, but was reversed and evidently improved by aeration conditions, which had the consistent trendsbetween the different treatments(Table 1). In addition, there were negligible differences in leaf chlorophyll content between the aeration and nonaeration conditions at 10 and 20 d after treatments (Table S2).

Effects of Cd and aeration on metal element accumulation

The contents of metal elements in the shoots and roots were determined after 20 d of different treatments. No significant differences were observed under aeration and nonaeration conditions with non-Cd stress, and significant differences were observed with 1.0 μmol/L Cd stress (Fig. 1-A and -B). However, the Cd contents of the two rice genotypes with 1.0 μmol/L Cd stress and aeration conditions were significantly higher than those under nonaeration conditions, increasing by 20.7% and 16.5% (roots), and 17.4% and 17.3% (shoots) of EJN1 and FP36, respectively. In addition, the Cd contents in the shoots of EJN1 were lower than those in FP36, with the opposite in roots. Therefore, it is suggested that the low Cd-accumulated rice (EJN1) had a lower capacity for Cd translocation from roots to shoots than the high Cd-accumulated rice (FP36). Furthermore, using a sequential extraction technique, the metal elements in two states (acid extractable and residual states) showed that the acid-extractable Cd (acid-Cd) in rice plants exhibited indistinctive differences between aeration and nonaeration conditions. However, with 1.0 μmol/L Cd stress, acid-Cd content in EJN1 and FP36 increased by 35.3% and 25.3% (roots), and33.8% and 24.5%(shoots) under aeration conditions, respectively (Fig. S2-A and -B). Moreover, the residual Cd (res-Cd) content under aeration conditions was also higher than that under nonaeration, especially at 1.0 μmol/L Cd stress, with up to 17.2% (EJN1) and 50.6% (FP36)in roots, and 45.8% (EJN1) and 6.1% (FP36)in shoots, respectively (Fig. S2-C and -D). These results suggested that the uptake and transport of Cd2+was markedly enhanced by roots under aeration conditions in rice plants.

Data are Mean ± SE (= 3). Different lowercase letters following the data within a column indicate the significance at< 0.05.

Moreover, the Fe content substantially decreased in EJN1 and FP36 under aeration conditions (Fig. 1-C and -D). Under non-Cd stress, the Fe contents in EJN1 and FP36were reduced by 21.3% and 41.4% (roots) and 16.1% and 11.6% (shoots), respectively, compared with nonaeration conditions. Under 1.0 μmol/L Cd stress, the Fe contents in EJN1 and FP36 were evidently decreased by 38.9% and 24.2% (roots) and 20.4% and 12.4% (shoots), respectively. In addition, the contents of acid-Fe and res-Fe in EJN1 and FP36 under aeration conditions were both significantly lower than those under nonaeration conditions (Fig. S2-E to -H). Thus, most of the Cd and Fe were distributed in an acid extractable state, and a few were in a residual state (Fig. S2). Similar to the accumulation of Cd and Fe in rice plants (Fig. 1), aeration conditions resulted in a significant increase of acid-/ res-Cd content in the two rice genotypes but a prominent decrease of acid-/res-Fe content, respectively (Fig. S2). Overall, under aeration conditions, an increase in Cd and a decrease in Fe accumulation occurred in rice roots and shoots.

Fig. 1. Cd and Fe contents in roots and shoots of Erjiunan 1 (EJN1) and Fupin 36 (FP36) at 20 d after two Cd treatments (0, 1.0 μmol/L) under aeration (O2) and nonaeration (Non) conditions.

Data are Mean ± SE (= 3). Different lowercase letters above the error bars indicate significant difference (< 0.05) among different treatments.

Effects of Cd and aeration on antioxidant enzymes

Superoxide dismutase (SOD) activity was suppressed byCd treatment andpromoted by the aeration treatment (Fig. 2-A and -B). With 1.0 μmol/L Cd stress, the SOD activities in the leaves of EJN1 and FP36 under aeration conditions were substantially enhanced by 55.0% and 87.8%, respectively, compared with those under nonaeration conditions (Fig. 2-B). The activities of peroxidase (POD) and catalase (CAT) differed between roots and leaves of both rice genotypes, and the POD activity in root was higher than that in leaf (Fig. 2-C and -D), whereas the CAT activity trend was reversed (Fig. 2-E and -F). Similar to the SOD activity, the POD and CAT activities in roots and leaves decreased with Cd treatment but improved with aeration treatment. The ascorbate peroxidase (APX) activity in roots and shoots was evidently induced by Cd and aeration treatments (Fig. 2-G and -H). In addition, with the same varying tendency of SOD, POD and CAT, the soluble protein content reached higher levels in leaves than in roots (Fig. S3-A and -B). In contrast, malonaldehyde (MDA) content increased with Cd levels and was more pronounced in FP36 than in EJN1, but aeration alleviated this effect in both roots and leaves (Fig. S3-C and -D).

Analysis of Fe transport-related gene expression

We used qRT-PCR to detect the expression of metal uptake/transport genes and investigate the effect of oxygen on Cd uptake and accumulation in rice plants. Rice, as an agraminaceous species, possesses a partial Strategy I system using the various Fe transporters as the components of the Fe2+uptake system, including,,and(Ishimaru et al, 2006; Kobayashi et al, 2014). The expression ofwas upregulated with Cd and aeration treatments(Fig. 3-A). Compared with those under nonaeration conditions, the expression ofunder aeration was significantly increased in the roots of EJN1 and FP36, especially with 1.0 μmol/L Cd stress (Fig. 3-B). Moreover, Cd stress induced the expression of, whose high expression resulted in an increase of Cd accumulation in roots, in accordance with previous reports by Takahashi et al (2011). The expression ofunder 1.0 μmol/L Cd stress was decreased compared with that under non-Cd stress (Fig. S4-A). Therefore,was affected by high Cd levels and aeration but was not the major gene mediating Cd uptake and accumulation in rice plants. Consequentlythe transcript levels ofandbut notwere strongly upregulated under Fe reduction due to Cd stress and aeration.

The expression ofin the roots of rice plants was significantly upregulated under aeration conditions. Especially with 1.0 μmol/L Cd stress, the expression was 4.30- and 7.05-fold higher in EJN1 and FP36 under aeration than their respective controls (Fig. 3-C). For, the increasing trend was in accordance with,andunder Cd and aeration treatments (Fig. 3). These results demonstrated that the expression of Fe-inducible genes (,,and) were significantly upregulated by aeration conditions. Cd exposure and aeration evidently decreased Fe accumulation to induce Fe reduction, which strongly upregulated the expression of Fe-inducible genes in rice plants.

Fig. 2. Activities of antioxidant enzymes in roots and leaves of Erjiunan 1 (EJN1) and Fupin 36 (FP36) at 20 d after two Cd treatments (0, 1.0 μmol/L) under aeration (O2) and nonaeration (Non) conditions.

SOD, Superoxide dismutase; POD, Peroxidase; CAT, Catalase; APX, Ascorbate peroxidase.

Data are Mean ± SE (= 3). Different lowercase letters above the error bars indicate significant difference (< 0.05) among different treatments.

Fig. 3. Expression of Fe-inducible genes (with the housekeeping geneas the internal control)in roots of Erjiunan 1 (EJN1) and Fupin 36 (FP36) at 20 dafter two Cd treatments (Cd0, 0 μmol/L; Cd1, 1.0 μmol/L) under aeration (O2) and nonaeration conditions.

The relative expression values were calculated using the 2-ΔΔCtmethod. Data are Mean ± SE (= 3). *,< 0.05; **,< 0.01.

displayed lower expression in the roots of the two rice genotypes and exhibited a decreasing trend under aeration conditions (Fig. S4-B). However, the expression ofin roots increased with Cd and aeration treatments (Fig. S4-C). In addition, root aeration resulted in an increase in the Zn content in the two rice genotypes (Fig. S5-A and -B), which was corresponded with the upregulation ofexpression. Therefore, our present investigation revealed that Cd stress and aeration controlled the expression levels ofandassociated with Cd and Zn accumulation in rice plants.had indistinctive differentunder different treatments in EJN1 rice plants, while it was significantly down- regulated by aeration treatment in FP36, compared to respective controls (Fig. S4-D). Moreover, the expressionlevels of two other Mn-specific transporters,and, were lower but slightly increased under aeration conditions (Fig. S4-E and -F). According to the elemental composition, Mn accumulation in differenttissues of the two rice genotypes significantly increased under aeration conditions regardless of Cd stress (Fig. S5-C and -D). Therefore, these results indicated that aeration had a negligible effect on the expression of Mn-related genes.

DISCUSSION

Root aeration affects physio-biochemistry and metal accumulation in rice

Cadmium pollution has become a serious problem worldwide, resulting in considerable agricultural productivity loss and heavy metal soil contamination (Ding et al, 2019).In rice production, intermittent/moist irrigation cultivation (such as aeration/oxygenation in hydroponics) is a well-known phenomenon that provides plenty of oxygen to farmlandsoil and is beneficial to rice root growth; furthermore, flooding/ waterlogging cultivationpatterns (such as hypoxic/ anoxic conditions) can alleviate the accumulation of Cd in soil. Therefore, the present study was carried out to determine the effects of aeration conditions in rhizospheres on the uptake and translocation of Cd in rice plants. Increasing Cd stress severely inhibited plant growth, which had also been obtained for other species, such as(Schutzendübel et al, 2001),(Ekmekci et al, 2008) and(Ghnaya et al, 2005). However, aeration can significantly reverse the effect of Cd-induced on the rice agronomic traits, such as increasing the plant height, root length, dry weight and root vigor (Table 1). Furthermore, Cd exposure causes oxidative stress by inducing the generation of ROS, which can be efficiently scavenged by antioxidative systems (including SOD, CAT, POD and APX) (Mittler et al, 2004; Romero-Puertas et al, 2004; Gill and Tuteja, 2010). In this study, Cd inhibited the activities of several antioxidant enzymes and induced lipid peroxidation reaction. However, these effects were significantly reversed by root aeration, which facilitated SOD, POD, CAT and APX activities and increased the soluble protein content in rice plants (Fig. 2 and Fig. S3),which is consistent with the results of Wang et al (2016). In contrast, the MDA content under Cd exposure was significantly elevated, and root aeration improved this effect. Interestingly, the APX activity increased under both Cd and aeration treatments, showing a positive correlation with the Cd content in both rice plants. Therefore, the high-level activities of antioxidant enzymes induced by aeration could protect plants against the oxidative damage and enhance the Cd tolerance of rice plants.

In addition, Cd interferes with the uptake and translocation of nutrient elements by plants, which negatively affects the ion homeostatic pathways, andreplaces the divalent cations(e.g., Fe2+, Mn2+and Zn2+)to transport into plant roots through ionchannel (Roth et al, 2006; DalCorso et al, 2008). In the present study, Cd exposure significantly reduced the Fe content, and aeration markedly accelerated this effect while also promoted Cd accumulation. Therefore, aeration couldaffect the uptake and accumulation of metallic elements in rice genotypes, especially under Cd stress, resulting in an increase and decrease in Cd and Fe contents, respectively, irrespective of the metal form (Figs. S1 and S2). Furthermore, the expression of Fe-inducible genes involved in Fe homeostasis is regulated by the demand for Fe acquisition (Bashir et al, 2013). Due to root aeration and Cd stress, Fe reduction could evidently upregulate the expression of Fe-inducible genes (e.g.,,,and). The effects of aeration on promoting Cd accumulation in rice were mediated by the regulation mechanisms of the Fe uptake-associated system. Chang et al (2020) found that the functions ofandin rice plants were similar and not redundant. However,contributed significantly to the uptake of Fe and Cd in roots, which was superior to the function ofin this study.

A gene regulatory network exists in aeration promotion of cadmium accumulation

On the one hand, oxygen plays a critical role in regulating the soil redox system by inducing an oxidative state in the soil around the roots, and the Fe2+/Fe3+redox system is a core factor responsible for the variation inredox potential. Fe mainly exists in the Fe3+state when exposed to oxygen but can rapidly reduce to Fe2+under hypoxic/anoxic conditions (Ding and Xu, 2011), which suggests that oxygen provides some signals related to the oxidation state in the rhizosphere of roots under aeration conditions, perhaps by changing the form or valence state of Fe in the rhizosphere. Moreover, rice plants have evolved intricate mechanisms classified as strategy I and II uptake systems to absorb Fe ion from soil. In the strategy I system, rice plants transport Fe2+through the Fe2+-transporters (such as OsIRT1 and OsNRAMP1) to across the root plasma membrane (Vert et al, 2002; Takahashi et al, 2011). Fe deficiency or reduction activates the Fe2+transport system to take up Fe from soil (Ishimaru et al, 2007). Rice also secretes small molecules (MAs) to bind Fe3+and form Fe3+-MA complexes that belong to the strategy II system, which can be readily absorbed by YSL (yellow stripe like) family transporters (e.g., OsYSL2, OsYSL15, OsYSL16 and OsYSL18) at the root surface (Koike et al, 2004; Aoyama et al, 2009; Inoue et al, 2009; Kakei et al, 2012). On the other hand, as a signal of the oxidation state, oxygen is probably a signalling molecule for the soil redox state and directly affects plant roots to regulate the expression of metal uptake/transport- related genes. However, roots can directly sense oxygen concentration in the rhizosphere, especially in response to hypoxia/anoxic conditions, such as through theanaerobic-responsive gene () and anaerobically inducible early gene in rice () (Dolferus et al, 1994; Huq and Hodges, 1999). Therefore, (in)sufficient oxygen markedly influences the expression of metal uptake genes (especially Fe-inducible genes) by altering the Fe2+/Fe3+redox system and ultimately affects the accumulation and morphology of Fe. Interestingly, root aeration significantlyelevated the expression ofto promote the Cd accumulation, and it could simulate the moist and intermittent irrigation during rice cultivation production. In contrast, hypoxic/anoxic conditions (such as flooded and waterlogged cultivation) can substantially decrease Cd uptake by downregulating the expression of(Chen et al, 2017). This explains why hydroponically cultured rice plants absorb less Cd than rice plants cultured in cyclical wet-dry environments and intermittent irrigation at the molecular level. In other words,participates in cellular Cd uptake and translocation, and high/low expression ofis a key factor for Cd accumulation in rice plants, which is easily influenced by the field environment. In addition, Mn exists in soils mostly as MnO2because of its low solubility in soil. There has been no recent report demonstrating the dissolution of MnO2to form Mn4+/Mn6+, but it can be reduced to form Mn2+and then stabilized in soil(Ding and Xu, 2011). Therefore, the effect of the redox system on Mn remains unclear. Moreover, this redox system is unlikely to have any effect on Zn, which maintains the divalent metal ions (Zn2+) in soil. In addition, aeration had no significant effect on the expression of Mn/Zn- related genes (,and), but the Mn/Zn contents in rice plants exhibited noticeable effects. Until recently, there has been no direct evidence showing that these Mn/Zn-related genes can transport Cd2+.

We speculated that due to their nonspecific substrate selectivity, divalent metal ion transporters are used to take up and transport Fe2+, Mn2+, Cd2+and Zn2+.The expression of metal transporter genes can be indirectly/directly influenced by other genes. For instance, the NAcontent was markedly enhanced under aeration regardless of Cd stress (Fig. S3-E and -F). NA is a structural analogue of MA that chelates metal cations (such as Fe, Mn and Zn). Thegene is reported to catalyze the formation of NA and participate in the MA biosynthesis pathway (Inoue et al, 2003). In the present study, upregulating the expression ofunder aeration in rice increased the accumulation of Zn and Mn to enhance tolerance to Fe reduction and attenuate the toxicity of excess Cd-stress (Fig. 4) (Lee et al, 2009b). Moreover, NA is converted into a 3′-keto intermediate via the transfer of an amino group by NA aminotransferase (Inoue et al, 2008) and is further converted to deoxymugineic acid (DMA). DMA is in combination with Fe3+to form Fe3+-DMA complex, then is transported by OsYSL15 from the rhizosphere to root and phloem (Lee et al, 2009a); therefore, a high NA content in rice plants induces high expression ofto maintain iron homeostasis. Furthermore, NA also affects the expression of, as an Mn-NA transporter, and regulates Mn accumulation in rice (Sasaki et al, 2011). Overall, oxygen affects the accumulation and translocation of Cd in rice plants via the Fe uptake process, and a gene regulatory network exists to control this complex pathway (Fig. 4).Furthermore,our findings indicated thatwas a crucial candidate for reducing the Cd accumulation in rice plants, and further experiments are necessary to verify this result. The present study provided a better understanding of different cultivation conditions (e.g., flooding and intermittent/moist irrigation) on Cd uptake, and the such information will be useful for reducing Cd accumulation in rice production.

Fig. 4. Model outlining of aeration promotes Cd accumulation due to Fe reduction in rice.

The yellow box represents the Cd-absorb/transport genes, the blue box represents the Fe-uptake/transport genes, and the grey box represents the Mn/Zn-transport genes.

METHODS

Rice materials and pretreatment conditions

Based on the Cd content in the grains, two earlyrice genotypes (procured from Jiaxing Institute of Agricultural Sciences, Zhejiang, China), Erjiunan 1 (EJN1, low Cd-accumulated rice) and Fupin 36 (FP36, high Cd-accumulated rice), were used and seeded in a sandy bed that had been previously rinsed with 0.05 mol/L H2SO4.At the four-leaf stage, uniform seedlings were selected and transplanted into 5.0-L plastic pots under hydroponic conditions and preincubated for 20 d. The nutrient solution contained the following concentrations of essential elements (mg/L): NH4NO3, 116.00; NaH2PO4·2H2O,49.90; K2SO4, 87.00; CaCl2, 111.00; MgSO4·7H2O, 418.00; MnCl2·4H2O, 1.80; (NH4)6Mo7O24·4H2O, 0.09; H3BO3, 1.10; ZnSO4·7H2O, 0.05; CuSO4·5H2O, 0.04; FeCl3·6H2O, 9.74; and citric acid (hydrate), 14.88. The solution was renewed every 3 d and adjusted to pH 5.5 with NaOH or HCl as required.

Cd stress and aeration treatments

After 20 d of preincubation, two Cd treatments (0and 1.0 μmol/L Cd supplied by CdSO4), and aeration [supplied by an ACO-5505 aquarium air pump (Hailea, Guangdong, China), with treatment conducted for 24 h] and nonaeration treatments were applied in hydroponics. The experiment was performed in a split-split plot design with four replicates, with the two rice genotypes as the main plots, two Cd levels as the subplots and aeration and nonaeration as the subsubplots.

Sampling and measurements under hydroponics

After 20 d of treatment, the rice plants were sampled to measure plant height, root length, plant dry weight (roots and shoots) and leaf chlorophyll content. The root samples were washed with 0.1 mol/L HCl several times and then washed with deionized water 3 times. The samples of both shoots and roots were dried at 110oC for 1 h and then dried at 65 oC in an oven to a constant weight (3 d). The dried samples were weighed, ground and used to determine metal element composition by an inductively coupled argon-plasma emission spectrometry (ICAP 61E trace analyser, Thermo-Jarrell Ashe, Franklin, MA, USA) and for the sequential extraction of different forms of metal elements from rice roots and shoots, i.e., the acid- extractable and residual forms.

The sequential extraction process was as follows: 0.50 g dry powder samples were soaked in 25 mL of 0.1 mol/L HCl at room temperature for 24 h and then centrifuged at 6 000 r/min for 10 min. The liquid supernatants were prepared as acid-extractable forms. After filtration through centrifugal filter units with a 0.22-μm hydrophilic PTFE membrane (Millipore, Billerica, MA, USA), the residues were prepared as the residual forms. The supernatants and residues were both analyzed using an ICAP 61E trace analyser (Thermo-Jarrell Ashe, Franklin, USA).

Assays of antioxidant enzyme activity, root vigor and NA content in rice plants

Root and leaf samples from EJN1 and FP36 for physiological and biological index detection were collected after 20 d of the different treatments. SOD, POD, CAT and APX activities, and soluble protein and MDA contents, as well as the root vigor (content of triphenyl tetrazolium chloride, TTC), of the two rice genotypes were determined using 0.50 g fresh roots, by the methods described by Zhao et al (2002). After treatment for 20 d, samples of roots and leaves were weighed (0.25 g) into a 10-mL centrifuge tube to determine the NA content, and 5 mL of water were added. After 1 min of eddy extraction, the volume was adjusted with water to 10 mL, and the sample was centrifuged at 9 500 r/min for 5 min. Next, the liquid supernatants were filtered using centrifugal filter units with a 0.22-μm hydrophilic PTFE membrane (Millipore, Billerica, USA). The filtered solutions were serially diluted with 20% liquid supernatant-purified water for NA content analysis, using LC-MS/MS (liquid chromatography-tandem mass spectrometry) which was conducted on a Waters Acquity UPLC/Quattro micro API (Yamaguchi and Uchida, 2012).

Gene expression analysis

Total RNA was extracted from the roots of EJN1 and FP36 at 20 d after the different treatments for qRT-PCR analysis of metal uptake/transport-related genes (,,,,,,,,and). The primer sequences for these specificgenes and(a housekeeping gene, as an internal control) are provided in Table S3. All analyses were performed at least three times, and the relative expression values were calculated using the 2-ΔΔCtmethod (Livak and Schmittgen, 2001).

Statistical analysis

The collected data were analyzed by three-factor variance analysis using a model specific for a split-split plot design, with the genotypes, Cd and aeration representing the main plot, subplot and subsubplot analyses, respectively. Three-way variance (ANOVA) and the least significant difference test (LSD) at< 0.05 were based on the Tukey’stest using SAS (SAS Institute, USA) (Smith et al, 2011).

ACKNOWLEDGEMENTS

This study was supported by the National Key Research and Development Plan of China (Grant No. 2017YFD0801102), the Central Public-Interest Scientific Institution Basal Research Fund, China(Grant No. 2017RG006-5), the National ScienceFoundation of China (Grant No. 31701407), and the Chinese Academy of Agricultural Sciences to the Scientific and Technical Innovation Team.

SUPPLEMENTAL DATA

The following materials are available in the online version of this article at http://www.sciencedirect.com/journal/rice-science; http://www.ricescience.org.

Fig. S1.Phenotypic characteristics of Erjiunan 1 and Fupin 36 after 20 d of different Cd stress and aeration treatments.

Fig. S2. Acid-/res-Cd and acid-/res-Fe contents in Erjiunan 1 and Fupin 36.

Fig. S3. Contents of soluble protein, malonaldehyde and nicotianamine.

Fig. S4. Expression of genesin roots of Erjiunan 1 and Fupin 36.

Fig. S5. Contents of Zn and Mn in roots and shoots of Erjiunan 1 and Fupin 36.

Table S1. Agronomic characteristics of Erjiunan 1 and Fupin 36 after 10 d of different Cd stress and aeration treatments.

Table S2. Contents of chlorophyll a, chlorophyll b, carotenoid and chlorophyll a/b of Erjiunan 1 and Fupin 36 after 10 d and 20 d of different treatments.

Table S3. Primer sequences for quantitative real-time PCR.

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2 August 2020;

17 November 2020

ShaoGuosheng (shaoguosheng@caas.cn)

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