Producing Cd-safe rice grains in moderately and seriously Cd-contaminated paddy soils

Highlights

• Different mitigation methods were compared in decreasing grain Cd in contaminated paddy soils.

• Liming/continuous flooding produced Cd-safe rice in soils with low degree of Cd contamination.

• Genetic engineering of rice plants was highly effective for the production of low Cd rice grain.

• Overexpression of OsHMA3 alone produced Cd-safe rice even in highly contaminated paddy soils.

Abstract

Rice grains produced on cadmium (Cd) contaminated paddy soils often exceed the maximum permissible limit. A number of mitigation methods have been proposed to decrease Cd accumulation in rice grain in contaminated acidic soils, including altering water management regimes, liming, and genetic engineering. In the present study, we conducted a pot experiment to compare these methods for their effectiveness at decreasing grain Cd concentrations in both acidic (pH 5.1–5.2) and alkaline (pH 7.5–7.9) paddy soils that varied in the degree of Cd contamination. In moderately Cd-contaminated acidic soils (with Cd concentrations lower than the intervention value of Chinese soil standard, GB15618-2018), any of the three methods was effective, reducing grain Cd concentration by 80–90% to levels below the Chinese maximum permissible limit (0.2 mg/kg). However, in the highly Cd-contaminated soils (with soil Cd concentrations exceeding the intervention value) with elevated concentrations of extractable Cd, although both liming and alternation of the water management regime (continuous flooding) was effective at decreasing grain Cd accumulation, grain Cd concentrations still exceeded the Chinese limit. Genetic engineering of rice, such as knockout of OsNramp5 (encoding the plasma membrane transporter responsible for Cd uptake into root cells) or overexpression of OsHMA3 (encoding a tonoplast Cd transporter sequestering Cd into the vacuoles), produced dramatic decreases (≥90%) in grain Cd concentration. Even in seriously contaminated soils, overexpression of OsHMA3 alone produced grain with Cd concentrations below the Chinese limit, offering a highly effective approach to produce Cd-safe rice especially in seriously Cd-contaminated paddy soils without affecting grain biomass or the concentrations of essential micronutrients.

Introduction

Cadmium is a Group I human carcinogen (IARC, 2018) that is ubiquitous in the environment (Smolders and Mertens, 2013). Rapid industrialization in many areas worldwide has resulted in widespread contamination of agricultural soils with Cd. In China, for example, it has been estimated that approximately 7% of the agricultural soils that were surveyed have been contaminated by Cd being as the contaminant of highest concern (MEE and MNR, 2014). In some areas of southern China, soil contamination has resulted in a considerable proportion of the rice (Oryza sativa) grain exceeding the Chinese maximum permissible limit for Cd concentration (0.2 mg/kg) (MHPRC, 2012). This broadscale Cd contamination has become a serious threat to food safety and human health (Zhao et al., 2015; Wang et al., 2019b; Zhao and Wang, 2020).

For the general non-smoking human population, the dominant pathway for Cd exposure is through dietary intake (Clemens et al., 2013). For populations that consume rice as their staple food, rice grains contribute more than 50% of the total dietary intake of Cd (Song et al., 2017; Wang et al., 2017). In some regions of southern China, the dietary intake of Cd exceeds the tolerable threshold (25 μg kg⁻¹ body weight) established by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) (Chen et al., 2018a). Long-term consumption of Cd-contaminated rice grain can cause serious human diseases, such as Itai-Itai disease, which occurred in the Jinzu river basin in Japan, renal dysfunction, osteoporosis, and increased risk of cancers (Nordberg et al., 2002, 2007, 2015; Jin et al., 2004). Therefore, reducing the accumulation of Cd in rice grain is a priority for ensuring food safety and protecting human health.

Cadmium has a high mobility within the soil-plant system and is relatively easily transferred from the soil to the grain (Zhao and Wang, 2020). However, several factors influence the availability of Cd within the soil-plant systems. The first important factor is the redox status of the soil. Rice paddies are generally flooded during the vegetative stages of rice growth but are then drained during the grain filling stage prior to harvest. During flooding, soil Cd is immobilized due to the formation of Cd sulfides (Cornu et al., 2007; Fulda et al., 2013; Hashimoto and Yamaguchi, 2013; Huang et al., 2013; Furuya et al., 2016). However, upon drainage, these Cd sulfides are readily oxidized, thereby releasing soluble Cd into soil porewater where it becomes available for plant uptake (Fulda et al., 2013; Furuya et al., 2016). Soil pH is another important factor determining Cd solubility in soils; an one-unit decrease in soil pH can lead to 3–4 fold increase in Cd solubility (Smolders and Mertens, 2013; Wang et al., 2019a). In acidic paddy soils, pH increases towards the near-neutral range when soils are flooded, resulting in a decrease in Cd solubility (Wang et al., 2019a). However, upon drainage, pH gradually decreases toward their initial (acidic) value, resulting in a marked increase in soluble Cd concentration (Smolders and Mertens, 2013; Wang et al., 2019a). This temporal pattern explains why more than 80–90% of the Cd that accumulates in the rice grain is derived from the mobilization of Cd in the soils when the paddy water is drained during the grain filling period (Inahara et al., 2007; Arao et al., 2009; Chen et al., 2020).

Once Cd is mobilized into the soil porewater, it is taken up into the root cells primarily through the Mn transporter, OsNRAMP5 (Natural Resistance-Associated Macrophage Protein 5) (Ishikawa et al., 2012; Sasaki et al., 2012). The OsNramp5 gene is predominantly expressed in rice roots with its protein polarly located at the distal side of the plasm membrane of the exodermis and endodermis, functioning as the major influx transporter for Mn and Cd into the root cells (Ishikawa et al., 2012; Sasaki et al., 2012). Compared to roots of wheat (Triticum aestivum) and maize (Zea mays), the influx of Cd into rice roots is greater due to a much higher expression level of Nramp5 in rice (Sui et al., 2018). Accordingly, knockout of OsNramp5 can markedly reduce the concentrations of Mn and Cd in the roots, shoots and grain (Sasaki et al., 2012; Yang et al., 2014; Tang et al., 2017). Once Cd is taken into the root cell, it can subsequently be sequestrated into the vacuoles by OsHMA3, a tonoplast transporter in the member of P_1B-type ATPase family (Ueno et al., 2010; Miyadate et al., 2011). OsHMA3 plays a crucial role in restricting the radial movement of Cd into the xylem and translocation to the above-ground tissues (Ueno et al., 2010; Miyadate et al., 2011). Some rice cultivars possessing weak or loss-of-function alleles of OsHMA3 have a decreased ability to sequester Cd into the vacuoles and consequently accumulate more Cd in the shoots and grain (Ueno et al., 2010; Miyadate et al., 2011; Yan et al., 2016; Sui et al., 2019). In contrast, overexpressing OsHMA3 in both Japonica and Indica cultivars can markedly decrease Cd accumulation in the grain (Ueno et al., 2010; Sasaki et al., 2014; Lu et al., 2019).

Two types of strategies have been proposed to reduce Cd accumulation in rice grain, which aim to reduce Cd availability in soil or to limit Cd uptake and/or translocation in plants (Zhao and Wang, 2020). For example, maintaining flooding condition or delaying drainage can decrease Cd availability and grain Cd concentrations (Bingham et al., 1976; Arao et al., 2009; Hu et al., 2013; Huang et al., 2013; Honma et al., 2016). Applications of liming materials to acidic paddy soils can also decrease Cd availability in the soil and Cd accumulation in rice grain (Zhu et al., 2016; Chen et al., 2018b). In terms of biological (genetic engineering) methods, knockout of OsNramp5 has been shown to be effective to reduce Cd uptake (Sasaki et al., 2012; Yang et al., 2014; Tang et al., 2017), whereas overexpression of OsHMA3 is highly effective at limiting Cd translocation (Ueno et al., 2010; Sasaki et al., 2014; Lu et al., 2019). However, the relative effectiveness of these methods has not been compared under the same conditions. It is also unclear whether different methods should be used simultaneously to deal with more heavily contaminated soils.

The present study aimed to compare the effectiveness of different mitigation strategies in both acidic and alkaline paddy soils, with the degree of Cd contamination varying from moderate to serious. Two contaminated alkaline soils were included as there is little information available regarding the effective methods to decrease Cd accumulation in rice grain in such soils.