Involvement of the Metallothionein gene OsMT2b in Drought and Cadmium Ions Stress in Rice

Abstract

Abiotic stress is one of the major factors restricting the production of rice (Oryza sativa L.). Developing rice varieties with dual abiotic stress tolerance is essential to ensure sustained rice production, which is necessary to illustrate the regulation mechanisms underlying dual stress tolerance. At present, only a few genes that regulate dual abiotic stress tolerance have been reported. In this study, we determined that the expression of OsMT2b was induced by both drought and Cd²⁺ stress. After stress treatment, OsMT2b-overexpression lines exhibited enhanced drought tolerance and better physiological performance in terms of relative water content and electrolyte leakage compared with wild-type (WT). Further analysis indicated that ROS levels were lower in OsMT2b-overexpression lines than in WT following stress treatment, suggesting that OsMT2b-overexpression lines had a stronger ability to scavenge ROS under stress. Reverse transcription-quantitative PCR (RT-qPCR) results demonstrated that under drought stress, OsMT2b influenced the expression of genes involved in ROS scavenging to enhance drought tolerance in rice. In addition, OsMT2b-overexpression plants displayed increased tolerance to Cd²⁺ stress, and physiological assessment results were consistent with the observed phenotypic improvements. Thus, enhancing ROS scavenging ability through OsMT2b overexpression is a novel strategy to boost rice tolerance to both drought and Cd²⁺ stress, offering a promising approach for developing rice germplasm with enhanced resistance to the abiotic stressors.

Introduction

Rice is a crucial crop globally, providing nutrition to nearly half of the world’s population. With rapid population growth, increasing grain yield has become the primary goal of rice breeding (Li et al. 2018). However, crops are frequently exposed to biotic and abiotic stresses, such as pests, pathogen infestations, precipitation, heat waves, and other extreme weather conditions, throughout their reproductive life, which substantially affect crop yield (Bailey-Serres et al. 2019). A considerable amount of water is required for rice cultivation. On average, 3,000–5,000 L of water are needed to produce one kilogram of rice (Yao et al. 2017). For instance, in China, rice cultivation consumes approximately 55% of the country’s freshwater resources (Luo et al. 2019). Statistics indicate that drought stress can reduce the yields of cereals, such as wheat, maize, rice, and barley, by approximately 40% (Rezaei et al. 2023). Thus, developing drought-resistant rice varieties is critical to overcome this challenge and ensure stable and improved rice yields (Hu and Xiong 2014).

After extensive evolution and selection, rice plants have developed multiple strategies to cope with drought stress. Research indicates that rice adapts to drought stress through morphological and physiological modifications, primarily via various signaling cascades and osmotic regulation (Hu and Xiong 2014). Numerous proteins, including transcription factors, protein kinases, and stress-related proteins, play roles in these adaptive processes. They enhance resistance to drought stress through various mechanisms, such as growth delay, transpiration reduction, osmotic regulation, and ROS scavenging (Liao et al. 2023; Xiang et al. 2007; Xu et al. 2008). Several genes associated with drought stress in rice have been identified and cloned using genetic and genomic approaches (Hu and Xiong 2014; Zhu et al. 2023). For instance, calcium-dependent protein kinase 9 (OsCPK9) has been reported to enhance osmoregulation and stomatal closure in rice under drought stress conditions (Wei et al. 2014). The reduction of stomatal density in epidermal patterning factor-like 10 (OsEPFL10) knockout lines reduces moisture loss, thereby improving drought tolerance in rice without affecting grain yield (Karavolias et al. 2023). Moreover, the overexpression of heat shock protein 50.2 (OsHSP50.2) in rice reduces cell membrane damage and lipid peroxidation under drought stress, enhancing superoxide dismutase (SOD) activity in response to drought (Xiang et al. 2018). Stress-responsive NAC 3 (ONAC003, SNAC3) positively regulates the expression of ROS-scavenging genes to maintain ROS homeostasis under drought stress, thereby enhancing drought tolerance (Fang et al. 2015). Furthermore, the overexpression of abscisic acid (ABA)-drought-reactive oxygen species 3 (OsADR3) in rice enhances sensitivity to exogenous ABA and increases the activities of various antioxidant enzymes, including catalase (CAT), glutathione peroxidase, and ascorbate peroxidase (APX), thus improving ROS scavenging capacity to positively regulate drought tolerance (Li et al. 2021).

Metallothioneins (MTs), a class of cysteine (Cys)-rich proteins, play a crucial role in abiotic stress as potent ROS scavengers and key components of nonenzymatic antioxidants (Dubey et al. 2019; Mekawy et al. 2020). MTs are also thought to participate in various metal tolerance or homeostasis processes since they can bind to metal ions (e.g., zinc (Zn), copper (Cu), and cadmium (Cd) through the abundant thiol groups found in their Cys residue (Hassinen et al. 2011). MTs are categorized into three major groups based on the number of Cys residues: Class I to III. Plant MTs belong to Class II and are further divided into four categories (MT1 to MT4) based on the number of Cys residues and their distribution at the N terminus. Each category exhibits distinct temporal expression patterns in various tissues and serves diverse functions (Saeed-Ur-Rahman et al. 2020). Several MTs have been cloned and characterized for their roles in rice. For example, OsMT-3a enhances tolerance to salt and heavy metal stress by scavenging ROS formed under these stresses (Mekawy et al. 2018). Furthermore, the overexpression of OsMT2c in Arabidopsis enhanced the ROS-scavenging ability of plants and enhanced their tolerance to Cu stress (Liu et al. 2015). In addition to heavy metals, plant MTs can respond to environmental stresses, such as drought, salt, and cold (Lee et al. 2004; Xue et al. 2009; Kumar et al. 2022). Overexpression of OsMT1a could substantially enhance the activities of CAT, peroxidase, and APX in rice, thereby improving the ROS-scavenging capacity and tolerance to drought stress (Yang et al. 2009). SEC13 Homolog 1 (OsSEH1) helps maintain OsMT2b levels by inhibiting its degradation mediated by the 26S proteasome, leading to decreased ROS levels and improved cold tolerance in rice (Gu et al. 2023). In addition, OsMT2b facilitates the accumulation and transport of zinc ions (Zn²⁺) from the plant to the grain through the phloem, alleviating Zn²⁺ stress (Lei et al. 2021). In epidermal rice cells, ethylene and H₂O₂ inhibited OsMT2b expression, thereby reducing ROS-scavenging capacity and increasing cell mortality (Steffens et al. 2009). Although studies have explored the function of OsMT2b in stress tolerance, its roles in drought and Cd²⁺ stress remain unclear. Therefore, in this study, we explored the function of OsMT2b in response to drought and Cd²⁺ stress. We determined that drought and Cd²⁺ stress induced OsMT2b expression. OsMT2b-overexpression lines exhibited increased tolerance to drought and Cd²⁺ stress. Furthermore, assays revealed that the ROS level was significantly lower in OsMT2b-overexpression lines than in WT. Our findings provide a basis for breeding rice with enhanced tolerance to drought and Cd²⁺ stress.

Results

Expression of OsMT2b is induced by drought and Cd ²⁺

We conducted RT-qPCR to investigate the expression pattern of OsMT2b under drought conditions. The results revealed that the expression of OsMT2b increased after 2 and 3 h polyethylene glycol (PEG) treatment (Fig. 1A). Because MTs are associated with heavy metal stress, we analyzed the expression of OsMT2b in rice seedlings exposed to CuSO₄, ZnSO₄, CdCl₂, AlCl₃, FeCl₃, and PbSO₄ through Northern blot analysis. The results indicated that CdCl₂ treatment upregulated OsMT2b expression (Fig. S1A). Subsequently, we observed that the expression of OsMT2b increased at 3, 4, 5, and 6 h after Cd²⁺ treatment (Fig. 1B). This finding indicated that PEG and Cd²⁺ treatment induced the expression of OsMT2b, implying its function in the responses to PEG and Cd²⁺.

Fig. 1

The expression of OsMT2b is induced by PEG and Cd²⁺. (A-B) The expression of OsMT2b in WT seedlings under PEG (A) and Cd²⁺ (B) treatment at different periods. The seedlings were cultured in a normal nutrient solution and then transferred to a nutrient solution containing 10% PEG (A) and containing 30 µM Cd²⁺ (B). The untreated seedlings were used as the control group. The leaves of seedlings were harvested separately at 0, 1, 2, 3, 4, 5, 6 and 8 h after no treatment, 10% PEG treatment and 30 µM CdCl₂ treatment. Values are presented as means ± SD (n = 3).

We fused the promoter region of OsMT2b with a β-glucosidase (GUS) reporter gene and then transformed the constructed proOsMT2b::GUS vector into the japonica variety ZH11. GUS staining results revealed prominent blue signals in the roots and germinal sheaths (Fig. S1B), suggesting that the OsMT2b gene is highly expressed in the roots and coleoptile.

Acquisition and detection of rice plants overexpressing OsMT2b

To investigate the function of OsMT2b, we constructed an overexpression vector driven by the Ubi promoter and obtained several OsMT2b-overexpression plants. Southern blot analysis performed using the Hygromycin phosphotransferase (HPT) selectable marker gene as a probe confirmed the successful integration of the exogenous gene into the rice genome, with most of the plants exhibiting a single-copy insertion (Fig. S2A). We selected two of these overexpression lines (OE22 and OE26) for a detailed examination. Phenotypic analysis revealed no significant differences in plant architecture between the overexpression lines and WT (Fig. S2B). The RT-qPCR assay demonstrated that the expression levels of OsMT2b in OE22 and OE26 were significantly higher than those in the WT (Fig. S2C). Moreover, Northern blot analysis revealed that the expression levels of OsMT2b in OE22 and OE26 were obvious higher than those in the WT (Fig. S3). These results indicated an enhanced expression of OsMT2b in transgenic plants.

Overexpression of OsMT2b increases tolerance to drought

To further investigate the function of OsMT2b, we subjected 2-week-old seedlings to drought stress simulated using 10% PEG. Before treatment, the growth patterns of overexpression and WT plants were similar (Fig. 2A). After 15 days drought treatment, WT plants exhibited leaf curling and clear dehydration symptoms, whereas the leaves of OE22 and OE26 displayed only mild signs of drought stress (Fig. 2B). After 15 days of recovery period, the majority of WT plants wilted and died, whereas the overexpression lines showed sprouting of new green leaves and resumed growth (Fig. 2C). The survival rates of OE22 and OE26 were 54.17% and 66.70%, respectively, which were significantly higher than that of the WT (8.30%) (Fig. 3A). These results suggested that the overexpression of OsMT2b increased drought tolerance in rice.

Fig. 2

Overexpression of OsMT2b positively regulates drought tolerance in rice. (A-C) The plant phenotype of WT and OsMT2b-overexpression plants before drought treatment (A), treated for 15 days (B), and recovered for 15 days (C). Scale bar: 5 cm. 2-week-old seedlings were treated in the nutrient solution containing 10% PEG for simulating drought treatment and transferred to a normal nutrient solution for recovery. WT: wild-type Zhonghua 11; OE22 and OE26: OsMT2b-overexpression lines.

Fig. 3

OsMT2b-overexpression plants exhibit increased drought tolerance. (A) The survival rate of WT and OsMT2b-overexpression lines after 15 d recovery. (B-C) The relative water content and electrolyte leakage of WT and OsMT2b-overexpression lines before and after drought treatment. WT: wild-type Zhonghua 11; OE22 and OE26: OsMT2b-overexpression lines. Values are presented as means ± SD (n = 3, * p < 0.05, *** p < 0.001; Student’s t-test).

To further verify that OsMT2b-overexpression plants exhibited increased drought tolerance, we measured the relative water content and electrolyte leakage in the leaves of rice seedlings. Before treatment, no significant difference in the relative water content was observed between OsMT2b-overexpression and WT plants (Fig. 3B). After 5 days of drought treatment, the relative water content in OE22 and OE26 was markedly higher than that in WT (Fig. 3B). Before treatment, no significant difference in electrolyte leakage was observed between overexpression and WT plants (Fig. 3C). After 5 days of drought treatment, electrolyte leakage was 27% in the WT but 5% and 6% in OE22 and OE26, respectively. Electrolyte leakage was significantly lower in OE22 and OE26 than in the WT (Fig. 3C). These results indicated that under drought stress, the leaves of WT were more severely damaged than those of OsMT2b-overexpression lines, confirming that the overexpression of OsMT2b enhances the tolerance of rice to drought stress.

Overexpression of OsMT2b enhances the ROS-scavenging capacity

MTs, known as ROS scavengers, are crucial for abiotic stress tolerance (Yang et al. 2009). We performed 3,3′-Diaminobenzidine (DAB) and nitro blue tetrazolium (NBT) staining to investigate ROS levels in WT and OsMT2b-overexpression plants. After 5 days of drought treatment, DAB staining revealed that the overexpression lines displayed a lighter color than the WT, especially in the second leaf, where WT plants exhibited significantly darker staining than the overexpression lines (Fig. S4A, B). Similar results were observed in the roots (Fig. S4C). These results demonstrated that hydrogen peroxide (H₂O₂) levels were lower in the overexpression lines than in the WT. NBT staining results also demonstrated that the WT displayed a more intense blue color than the overexpression lines (Fig. S4D, E), suggesting that OsMT2b-overexpression lines possess a stronger ability to scavenge ROS under drought stress.

We measured H₂O₂ levels in the leaves of the plants before and after drought treatment. The results revealed that H₂O₂ levels were significantly lower in the leaves of overexpression plants than in those of WT plants before drought treatment. After drought treatment, the H₂O₂ level tended to increase in leaves over time in all plants, and it was consistently significantly lower in overexpression plants than in WT plants (Fig. 4A). We also measured the H₂O₂ level in the roots of the plants and determined that the H₂O₂ level was consistently significantly lower in the roots of overexpression plants than in those of WT plants (Fig. 4B). These results suggested that overexpression of OsMT2b enhanced the ROS-scavenging ability.

Fig. 4

OsMT2b positively regulates H₂O₂ scavenging capacity in overexpression lines. (A-B) H₂O₂ concentrations in leaves (A) and roots (B) of WT and OsMT2b-overexpression lines were determined under normal and drought stress treatments. The leaves and roots of seedlings were collected separately at 0, 1, 3, and 5 d after 10% PEG treatment. WT: wild-type Zhonghua 11; OE22 and OE26: OsMT2b-overexpression lines. Values are presented as means ± SD (n = 3, * p < 0.05, ** p < 0.01; Student’s t-test).

Overexpression of OsMT2b enhances Cd ²⁺ tolerance

Most MTs can chelate heavy metal ions, and CdCl₂ treatment induced the expression of OsMT2b (Fig. S1A). To examine the response of overexpression plants to Cd²⁺ stress, we treated 2-week-old seedlings with 30 µM CdCl₂. Before treatment, the growth pattern of WT and overexpression plants was found to be similar (Fig. 5A). After 15 days of treatment, WT plants exhibited leaf curling and wilting, whereas OE22 and OE26 plants were less affected and maintained basal growth status (Fig. 5B, Fig. S5). Furthermore, no significant difference in plant height was observed between the WT and overexpression plants before treatment. However, overexpression plants were significantly taller than WT plants after Cd²⁺ treatment (Figs. 5C and 6A).

Fig. 5

Overexpression of OsMT2b positively regulates Cd²⁺ tolerance in rice seedlings. (A-C) The phenotype of WT and OsMT2b-overexpression lines before Cd²⁺ treatment (A), treated for 15 days (B), and recovered for 15 days (C). Scale bars: 5 cm. 2-week-old seedlings were treated in the nutrient solution containing 30 µM CdCl₂ and transferred to a normal nutrient solution for recovery. WT: wild-type Zhonghua 11; OE22 and OE26: OsMT2b-overexpression lines

Fig. 6

Overexpression of OsMT2b enhances Cd²⁺ tolerance. (A-D) The plant height (A), electrolyte leakage (B), fresh seedling weight (C), and dry seedling weight (D) of WT and OsMT2b-overexpression lines before and after Cd²⁺ treatment. WT: wild-type Zhonghua 11; OE22 and OE26: OsMT2b-overexpression lines. Values are presented as means ± SD (n = 3, * p < 0.05, ** p < 0.01; Student’s t-test).

To examine the tolerance of OsMT2b-overexpression plants to Cd²⁺, we measured the physiological indices of seedlings before and after Cd²⁺ treatment. Before Cd²⁺ treatment, no significant difference in electrolyte leakage was observed between the WT and overexpression lines. After Cd²⁺ treatment, electrolyte leakage in overexpression plants was markedly lower than that in WT plants (Fig. 6B). Moreover, no significant difference in fresh weight was observed between the overexpression and WT plants before treatment. After Cd²⁺ treatment, the fresh weight of overexpression plants was considerably higher than that of WT plants (Fig. 6C). The dry weight of overexpression plants did not markedly differ from that of WT plants without Cd²⁺ treatment. While after Cd²⁺ treatment, the dry weight of overexpression plants was significantly higher than that of WT (Fig. 6D). These results indicated that the overexpression plants experienced less damage than WT plants after Cd²⁺ treatment, suggesting that the overexpression of OsMT2b improved the tolerance of rice to Cd²⁺ stress.

To determine the reasons for increased tolerance to Cd²⁺ stress in OsMT2b-overexpression plants, we performed NBT staining. The results revealed no difference between the leaves of WT and overexpression plants before Cd²⁺ treatment. The leaves of both WT and overexpression plants displayed deeper staining after 6 h of treatment than before treatment, indicating that Cd²⁺ treatment increased the superoxide anion levels in leaves. In addition, staining in the OE22 and OE26 leaves was lighter than that in WT (Fig. S6). These results indicated that the accumulation of superoxide anions was significantly lower in OsMT2b-overexpression plants than in WT plants.

OsMT2b Influences the Expression of Several drought-resistance Genes

To further explore the role of OsMT2b in drought stress response, we examined the expression levels of several drought-responsive genes under normal and drought conditions. Basic leucine zipper 23 (OsbZIP23), osmotic stress/ABA–activated protein kinase 3 (OsSAPK3), and myo-inositol oxygenase (OsMIOX) positively regulate drought tolerance in rice (Xiang et al. 2008; Lou et al. 2023; Duan et al. 2012). The results revealed no significant difference in the expression levels of OsbZIP23 and OsMIOX between OsMT2b-overexpression and WT plants under normal conditions. OsSAPK3 levels were decreased in overexpression lines. After 48 h of drought treatment, the expression levels of OsbZIP23, OsSAPK3, and OsMIOX were significantly higher in the overexpression lines than in WT lines (Fig. 7). This finding suggested that OsMT2b promotes the expression of these drought-responsive genes under drought conditions.

Fig. 7

OsMT2b influences the expression of several drought-resistant genes. The expression levels of OsbZIP23 (A), OsSAPK3 (B) , OsMIOX (C) , DSM1 (D) , OsSKL2 (E) , OsLG3 (F) , OsDRZ1 (G) , OsAAI1 (H) and SNAC3 (I) in WT and OsMT2b-overexpression lines before and after drought treatment for 48 h. The seedlings were cultured in a normal nutrient solution and then transferred to a nutrient solution containing 10% PEG. The leaves of seedlings were collected before and after drought treatment for 48 h. WT: wild-type Zhonghua 11; OE22 and OE26: OsMT2b-overexpression lines. Values are presented as means ± SD (n = 3).

We examined changes in the expression levels of drought-hypersensitive mutant 1 (DSM1), shikimate kinase-like 2 (OsSKL2), ethylene-responsive factor 62 (OsERF62; OsLG3), drought-responsive zinc finger protein 1 (OsDRZ1), Alpha-amylase inhibitor 1 (OsAAI1), and SNAC3 between the WT and OsMT2b-overexpression plants under normal and drought conditions. These genes enhanced drought tolerance by mediating ROS scavenging (Ning et al. 2010; Fang et al. 2015; Xiong et al. 2018; Yuan et al. 2018; Jiang et al. 2022; Long et al. 2023). Under normal conditions, the expression levels of DSM1, OsSKL2, and OsLG3 did not significantly differ between OsMT2b-overexpression lines and WT. The expression levels of OsDRZ1, OsAAI1, and SNAC3 were downregulated in the OsMT2b-overexpression lines. After 48 h of drought treatment, the expression levels of DSM1, OsSKL2, OsLG3, and OsDRZ1 were significantly upregulated in the overexpression lines. However, no changes were noted in the expression of OsAAI1 and SNAC3 (Fig. 7). These results indicated that OsMT2b enhances drought tolerance in rice by regulating the expression of drought-responsive genes involved in ROS scavenging.

Discussion

MTs, a family of Cys-rich proteins, play a crucial role in maintaining metal ion homeostasis in plants by chelating metal ions. They are extensively involved in regulating stress responses in plants and possess the ability to scavenge free radicals (Zhu et al. 2010). Previous studies have demonstrated that OsMT2b exerts a positive regulatory effect on pathogen resistance and cold tolerance in rice (Wong et al. 2004; Gu et al. 2023; Lei et al. 2021). In this study, we determined that drought stress and exposure to the heavy metal Cd²⁺ induced the expression of OsMT2b, which is consistent with the published results. Exposure to copper upregulated the expression of OsMT2c, a scavenger of ROS, by inducing H₂O₂ generation (Liu et al. 2015). The expression of OsMT1e was significantly induced under Cd²⁺ stress (Rono et al. 2021). CtMT1 in guar (Cyamopsis tetragonolobus L.) is a homologous gene of OsMT2b. The expression of CtMT1 was upregulated in leaves and roots under drought stress (Jaiswal et al. 2018). Furthermore, the expression of the MT gene GhMT3a was induced by drought stress in cotton. In addition, the ectopic expression of GhMT3a in tobacco significantly reduced the ROS level. Compared with WT plants, tobacco plants with GhMT3a overexpression exhibited significantly improved drought tolerance (Xue et al. 2009). These findings indicate that OsMT2b may play a role in enhancing tolerance of rice to Cd²⁺ and drought stress.

Furthermore, our results indicated that OsMT2b-overexpression lines exhibited better growth and survival rates under drought treatment, which is consistent with the finding indicating improved drought tolerance in rice plants by overexpressing OsMT1a (Yang et al. 2009). Overexpression of GhMT3a in tobacco also improved tolerance to salt, drought, and cold stresses (Xue et al. 2009). According to these researches, MT genes may confer resistance to more than one type of stress. In this study, we observed that OsMT2b-overexpression plants improved tolerance to Cd²⁺ stress, with an increase in plant height and relative water content and a decrease in electrolyte leakage. The rice homologous genes OsMTI-1b and OsMTII-1a can bind to Cd²⁺ (Nezhad et al. 2013). OsMT-3a plays a role in the chelation of Cd²⁺, thereby increasing Cd²⁺ stress tolerance in E. coli cells and rice plants (Mekawy et al. 2018, 2020). Overexpression of the Arabidopsis MT gene MT3 in Vicia faba increased tolerance to Cd²⁺ stress (Lee et al. 2004). These findings align with our results, indicating the potential applications of this gene in enhancing resistance to drought and Cd²⁺ stress.

ROS plays a crucial role in plant responses to biotic and abiotic stresses (Baxter et al. 2014). These highly reactive and toxic molecules can lead to lipid peroxidation and cell membrane damage, with their levels indicating the extent of cellular damage (Miller et al. 2010). The antioxidant properties of MTs are largely due to their high Cys content, enabling them to bind metal ions and scavenge ROS (Hassinen et al. 2011). In this study, we determined that, following DAB and NBT staining, the coloration in OsMT2b-overexpression plants was lighter than that in WT plants under drought or Cd²⁺ treatment. This finding suggests that the overexpression of OsMT2b scavenges excessive ROS generated by external environmental conditions, thereby preventing cell damage caused by ROS accumulation. It is possible that the lower level of ROS in OsMT2b-overexpression plants is due to the decrease of active Cd²⁺ caused by the binding between OsMT2b and Cd. Previous studies have demonstrated that the overexpression of SNAC3 in rice modulates ROS homeostasis by regulating the expression of ROS-scavenging genes, thereby enhancing drought and heat tolerance (Fang et al. 2015). Similarly, OsMT1a enhanced drought tolerance in rice by participating in the ROS-scavenging pathway and maintaining Zn²⁺ homeostasis (Yang et al. 2009). OsMTI-1b and OsMT-3a improved metal ion tolerance by reducing ROS levels (Malekzadeh et al. 2017; Mekawy et al. 2020). Thus, the improved tolerance to drought and Cd²⁺ stress in OsMT2b-overexpression plants can be attributed to their ROS-scavenging capacity. In addition, changes in the ROS levels in plants led to the altered expression of various ROS-related genes, further stabilizing ROS balance.

Conclusions

Our results demonstrated that OsMT2b positively regulates rice tolerance of drought and Cd²⁺ by enhancing their ROS-scavenging capacity. This study deepens our understanding of the function of metallothionein gene OsMT2b in rice and illustrates the regulation mechanisms of OsMT2b in responding to drought and Cd²⁺ stress. This is helpful to breeders of improving crop tolerance to abiotic stresses, like drought and Cd²⁺.

Methods

Plant Materials and Growth Conditions

All experiments were carried out using the rice variety Zhonghua 11 (Oryza sativa L. ssp. japonica cv.). The WT and transgenic plants were grown in a paddy field at South China Agricultural University in Guangzhou.

Drought and Cadmium Chloride Treatments

For drought stress conditions, WT and overexpression lines were transferred to Kimura B complete nutrient solution (Yoshida et al. 1976) after germination for 4 days. When the seedlings were 2 weeks old, they were treated with a new Kimura B complete nutrient solution containing 10% PEG to simulate drought treatment and maintained in a chamber with a 13 h light/11 h dark cycle. The plants were photographed before treatment. After 15 days of drought treatment, we transferred the seedlings to a new Kimura B complete nutrient solution for 15 days of recovery. After recovery, the survival rate of seedlings was counted, and the relative water content and electrolyte leakage were measured before treatment and after treatment for 5 days. 25-day-old seedlings treated with 10% PEG were collected separately at 0, 1, 2, 3, 4, 5, 6 and 8 h and stored at -80 °C for RNA extraction. The untreated seedlings were used as the control group.

For CdCl₂ treatment, 2-week-old seedlings were transferred to Kimura B complete nutrient solution containing 30 µM CdCl₂ and maintained in a chamber with a 13 h light/11 h dark cycle. The plants were photographed before treatment. After 15 days of Cd²⁺ treatment, the seedlings were transferred to a new Kimura B complete nutrient solution for 15 days of recovery. The electrolyte leakage, plant height, fresh seedling weight and dry seedling weight was measured before and after treatment. 25-day-old seedlings treated with 30 µM CdCl₂ were collected separately at 0, 1, 2, 3, 4, 5, 6 and 8 h for RNA extraction. The untreated seedlings were used as the control group.

RNA Extraction and RT-qPCR

Each sample was ground in liquid nitrogen with a mortar, then total RNA was extracted with TRIzol reagent (Takara) and quantified with a DU730 spectrophotometer (Beckman Coulter, Germany). cDNA was synthesized using a StarScript III RT Kit (Genstar). RT-qPCR was conducted on the Bio-Rad CFX 96 using SYBR premix Ex Taq II following the manufacturer’s protocol. Rice UBIQUITIN (UBI, LOC_Os03g13170) gene was used as internal reference, and relative quantification was used for data analysis. All primers for RT-qPCR were listed in Table S1.

Northern Blot Analysis

The extracted total RNA was separated by electrophoresis on 1.5% denaturing agarose gels, then transferred to hybond-N⁺ membranes (Amersham Bioscience, Buckinghamshire, UK) and the membrane was hybridized overnight with a probe labeled with digoxigenin (DIG) based on the OsMT2b gene sequences in a hybridization buffer at 42 °C, as described previously (Jiang et al. 2009). Hybridization signals were detected using CDP-star and visualized using a ImageQuant 400 imaging system (GE Healthcare, Piscataway, NJ, USA).

Southern blot Analysis

To determine the copy number of T-DNA insertion in transgenic plants, Southern blot analysis was performed using a HPT specific fragment probe. HPT fragment was amplified using the HPT-F and HPT-R primers (Table S1). The Southern bolt procedures were performed as described previously (Ni et al. 2018). Briefly, approximately 7 µg genomic DNA of each sample was digested, and the following separated by electrophoresis on a 0.8% agarose gel, then transferred to a nylon membrane, hybridization in a hybridization buffer.

GUS Histochemical Staining

To characterize the expression patterns of OsMT2b, proOsMT2b::GUS transgenic rice plant was created. Seedlings of proOsMT2b::GUS transgenic plants were fixed for 30 min in a 90% (v/v) acetone, vacuumed for 15 min and then placed in an ice bath for 30 min. The fixed tissues were washed three times in 100 mM sodium phosphate buffer containing 0.1% (v/v) Triton X-100, 1 mM potassium ferricyanide and 1 mM potassium ferrocyanide. The above tissues were subsequently stained in a 100 mM sodium phosphate buffer, pH 7.0, containing 0.1% (v/v) Triton X-100, 0.5 mg/mL X-Gluc, 1 mM potassium ferricyanide and 1 mM potassium ferrocyanide at 37 °C overnight after being vacuumed for 10 min. After histochemical staining, the green-tissue samples were immersed in 70% ethanol for 1 day to remove chlorophyll and observed under a microscope or camera.

ROS Level Measurements

The seedlings were harvested at 5 days after drought stress treatment and fully expanded leaves were collected at 0, 6 and 48 h after Cd²⁺ treatment for NBT staining. Then these samples were incubated in potassium-citrate buffer (10 mM, pH 6.0) and placed under a vacuum for 10 min. Later they were placed in potassium-citrate buffer containing 0.5 mM NBT for 3 h (dark, 25 °C).

For DAB staining, the procedures were performed as described previously (Zheng et al. 2021). The seedlings were collected after drought stress treatment and incubated with 0.1% (w/v) DAB (pH 3.8) for 2 h in a growth chamber. Subsequently, the leaves were immersed twice with 70% ethanol and photographed with a camera.

The formation of H₂O₂ was quantitatively analyzed with a hydrogen peroxide assay kit (#S0038, Beyotime Biotech, Shanghai, China) according to the manufacturer’s protocol. Seedlings were treated with 10% PEG for 0, 1, 3, and 5 days, and then approximately 0.1 g of leaves or roots were ground in liquid nitrogen. Afterward, the powder was extracted from 1 mL of extraction buffer. After centrifugation (12,000 g, 15 min, 4 °C), the supernatant was used to determine H₂O₂ levels by the spectrophotometer method. All measurements were conducted three times and analyzed with Student’s t-test.

Measurement of Relative Water Content

To measure the relative water content, 3–6 fully expanded leaves of each line without treatment and after treatment for 5 days were collected. Then, the fresh weight was recorded as M1. The leaves were soaked in tap water for 2 h in a 50 mL centrifuge tube and quickly weighed and recorded as M2. Subsequently, the leaves were placed in a drying oven at 60 °C for 10–15 h, until no moisture was lost and weighed their dry weight and recorded as M3. Finally, calculation according to the formula: Relative water content (%) = (M1-M3)/(M2-M3) × 100.

Electrolyte Leakage Assay

To measure the electrolyte leakage, 3 cm long sections from the middle of the leaf blades were collected. These samples were rinsed 3 times with deionized water and drained with absorbent paper. Then the samples were placed in 15 mL centrifuge tubes containing 5 mL of deionized water and incubated at room temperature (RT) until the ions are fully released. A conductance meter (DDS-11 A, Hongyi Instrument CO., Ltd, Shanghai, China) was used to measure the electrical conductance in each tube of the solution and the electrical conductance value was recorded as M0. Subsequently, the samples were incubated at RT for 24 h and the detected electrical conductance value was recorded as M1. The samples were placed in boiling water for 30 min, then equilibrated to RT, the detected electrical conductance value was recorded as M2. The electrolyte leakage (EL) was expressed as EL (%) = (M1-M0)/(M2-M0) × 100.