Physiological and Transcriptomic Responses of Rice Cultivars to Combined Cadmium and Elevated Temperature Stress

TL;DR

This study explores how two rice varieties respond to cadmium contamination and higher temperatures, revealing different strategies for growth and stress tolerance.

Contribution

The study identifies genotype-specific physiological and transcriptomic responses to combined cadmium and temperature stress in rice.

Findings

YZX showed greater growth increases at higher temperatures but accumulated more cadmium in roots.

XWX12 exhibited stronger antioxidant enzyme activity and reduced cadmium accumulation in shoots.

Transcriptomic analysis revealed distinct strategies for Cd sequestration and stress response between the two cultivars.

Abstract

Cadmium (Cd) contamination and rising temperatures pose significant challenges to rice growth and food safety. Here, we investigated growth responses, Cd accumulation, physiological adaptations, and transcriptomic profiles of two rice cultivars, Yuzhenxiang (YZX) and Xiangwanxian 12 (XWX12), under combined Cd (0, 5, 20 μmol L−1) and temperature (25 °C, 30 °C) stress. Moderate warming (30 °C) generally promoted seedling growth and enhanced Cd uptake, with YZX showing greater increases in plant height and biomass, whereas XWX12 developed longer roots. At maturity, the temperature-induced growth advantage persisted in YZX, accompanied by a 60% increase in root Cd concentration, compared with 36% in XWX12. Antioxidant enzyme activities (POD, SOD, CAT) were significantly induced under combined stress, with XWX12 exhibiting stronger enzymatic responses and broader activation of ABC transporter genes, supporting reduced Cd accumulation in shoots. Malondialdehyde content indicated milder oxidative damage in YZX despite higher Cd accumulation. Transcriptomic analyses revealed extensive early transcriptional reprogramming, with enrichment of antioxidant metabolism, ABC transporters, MAPK signaling, and Cd transport-related genes, demonstrating coordinated physiological and molecular responses. XWX12 favored intracellular Cd sequestration and sustained antioxidant activation, whereas YZX relied more on uptake and translocation processes. Overall, these results highlight genotype-specific strategies in coping with combined Cd and temperature stress, providing mechanistic insights for improving rice tolerance and safety under warming and contaminated environments.

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1. Introduction

Cadmium (Cd) is one of the most hazardous heavy metal pollutants in agricultural ecosystems due to its high toxicity, mobility, and persistence [1,2]. In rice-based cropping systems, Cd readily migrates along the soil–rhizosphere–plant continuum and accumulates in grains, often at higher levels than other heavy metals [3,4]. Long-term consumption of Cd-contaminated rice poses serious health risks, making Cd pollution a major constraint on food safety and sustainable agriculture, particularly in regions where rice is a dietary staple [5,6].

Rice (Oryza sativa L.) exhibits pronounced genotypic variation in Cd accumulation, with indica cultivars generally accumulating higher grain Cd concentrations than japonica cultivars [7]. This divergence is closely associated with the genetic regulation of Cd uptake by roots, long-distance transport, and intracellular sequestration [8]. Cd stress inhibits seed germination, root growth, and photosynthetic performance, disrupts mineral nutrient homeostasis, and ultimately reduces yield and grain quality [9].

At the molecular level, Cd uptake, transport, and detoxification in rice are regulated by multiple metal transporters [10]. OsNRAMP5 functions as the primary Cd influx transporter in roots and is a key determinant of Cd acquisition [11,12]. In contrast, the vacuole-localized P1B-type ATPase OsHMA3 limits long-distance Cd translocation by sequestering Cd in root cell vacuoles. Loss-of-function or weak OsHMA3 alleles, which are common in certain indica cultivars, represent an important genetic basis for high grain Cd accumulation [13,14,15]. In addition, ZIP family members, ABC transporters, and pleiotropic drug resistance proteins contribute to Cd redistribution and detoxification, while Cd-related genes expressed in vascular tissues influence xylem loading and root–shoot–grain translocation efficiency [16,17].

Cd accumulation is not solely genetically controlled but is also highly responsive to environmental conditions [18]. Temperature has recently emerged as an important factor influencing Cd dynamics; elevated temperature of 30 °C can enhance root metabolic activity and membrane transport processes, promoting Cd uptake and internal translocation, with stronger effects observed in indica rice [19,20]. However, under natural conditions, Cd and moderate warming frequently co-occur as combined stresses, and their interactive effects on Cd transport, intracellular sequestration, and genotypic variation remain poorly understood.

In this study, two late-season rice cultivars with contrasting Cd accumulation capacities, a high-Cd-accumulating indica cultivar (Yuzhenxiang) and a low-Cd-accumulating japonica cultivar (Xiangwanxian 12), were used to investigate responses to combined Cd and temperature stresses. Growth traits, photosynthetic performance, antioxidant responses, and transcriptomic profiles were systematically compared. By integrating physiological and transcriptomic analyses, this study aims to elucidate genotype-dependent regulatory patterns of Cd-related transport and detoxification genes under combined environmental stresses.

2. Results

2.1. Growth Responses and Cadmium Accumulation of Rice Seedlings Under Combined Cadmium and Temperature Stress

The interaction between cadmium (Cd) stress and elevated temperature significantly affected seedling growth and Cd accumulation in rice, with pronounced cultivar-dependent differences [21]. Under combined Cd (0, 5, and 20 μmol L^−1^) and temperature (25 °C and 30 °C) treatments, Yuzhenxiang (YZX) and Xiangwanxian 12 (XWX12) exhibited distinct responses in plant height, root length, and biomass accumulation. Overall, moderate warming (30 °C) promoted seedling growth, whereas increasing Cd concentration progressively attenuated this stimulatory effect. Under 30 °C and low to moderate Cd levels (0–5 μmol L^−1^), YZX showed a more pronounced increase in plant height and fresh weight, whereas XWX12 consistently developed longer roots across all Cd concentrations, indicating a relative advantage in root growth. However, at 20 μmol L^−1^ Cd, both cultivars exhibited significant reductions in plant height, root length, and fresh weight, and the growth advantage of YZX observed under lower Cd levels was abolished, suggesting that severe Cd stress overrides the growth-promoting effect of elevated temperature (Figure 1A–F). In addition, both cultivars displayed higher root-to-shoot ratios at 30 °C, implying enhanced biomass allocation to belowground organs, while high Cd exposure constrained overall biomass accumulation.

Consistent with these growth responses, Cd accumulation and internal redistribution in rice seedlings were strongly influenced by both temperature and genotype (Figure 2A–C). Under treatment at 30 °C, generally increased Cd accumulation in roots, stems, and leaves of both cultivars, although their response patterns differed markedly. Across all treatments, YZX accumulated higher Cd levels than XWX12, with Cd predominantly retained in roots, followed by stems and leaves. At 25 °C, Cd content in YZX roots increased significantly with rising external Cd supply, whereas Cd levels in stems and leaves remained relatively stable. In contrast, XWX12 exhibited pronounced increases in Cd accumulation in stems and leaves, with little change in root Cd content. Under 30 °C, Cd concentrations in both roots and leaves of YZX increased significantly, whereas XWX12 showed a coordinated increase in Cd content across all organs under high Cd exposure, indicating greater sensitivity to combined Cd and heat stress. Notably, the Cd translocation factor was significantly higher in YZX than in XWX12 and increased by 81% and 64% under high Cd conditions, respectively, whereas XWX12 exhibited enhanced translocation efficiency only at elevated Cd levels (Figure 2D). These results demonstrate that moderate warming facilitates Cd uptake and internal redistribution, and that YZX possesses a stronger capacity for Cd accumulation and translocation, highlighting distinct adaptive strategies among rice cultivars under combined metal and thermal stress.

2.2. Effects of 30 °C on Plant Height and Cadmium Accumulation at the Maturity Stage

To determine whether the temperature-dependent responses observed at the seedling stage persist into later developmental stages, we further examined plant height and Cd accumulation in rice at maturity under identical Cd-contaminated soil conditions. Compared with 25 °C, exposure to 30 °C significantly increased plant height in both cultivars, although the magnitude of response differed between genotypes (Figure 3A). Specifically, the plant height of YZX increased by 32.1%, whereas the increase in the XWX12 was not significant, indicating a stronger growth response to 30 °C in YZX (Figure 3B).

Consistent with the enhanced vegetative growth, 30 °C also markedly promoted Cd accumulation at the maturity stage. Root Cd concentration increased by 60.2% in YZX and by 36.0% in XWX12 under 30 °C. Notably, Cd accumulation in aboveground tissues exhibited a pronounced cultivar-dependent pattern: YZX exhibited a significant increase in cadmium content in the shoot, reaching 9.4%, whereas XWX12 showed only a slight elevation in shoot (Figure 3C). These results demonstrate that exposure to 30 °C not only stimulates plant growth at maturity but also enhances Cd uptake and translocation, particularly in YZX, further supporting the persistence of genotype-specific responses to combined Cd stress and temperature elevation throughout rice development.

2.3. Antioxidant Enzyme Responses and Oxidative Damage in Rice Seedlings Under Combined Cadmium and Temperature Stress

Antioxidant enzyme activities in rice seedlings were markedly altered, reflecting the growth inhibition and increased Cd accumulation induced by combined Cd and temperature stress. reflecting cultivar-specific physiological adjustments to abiotic stress ((Figure 4A–D)). Peroxidase (POD) activity increased with rising Cd concentration and temperature in both cultivars. At 25 °C, POD activity in YZX remained unchanged at 5 μmol L^−1^ Cd but increased by 17.2% at 20 μmol L^−1^ Cd, whereas XWX12 showed pronounced increases of 46.5% and 85.4% at 5 and 20 μmol L^−1^ Cd, respectively. At 30 °C, both cultivars exhibited significant POD activation under high Cd stress (20 μmol L^−1^), with increases of 39.2% in YZX and 36.8% in XWX12 (Figure 4A). Superoxide dismutase (SOD) activity displayed a pattern similar to that of POD. In YZX, SOD activity increased significantly at all Cd concentrations, with a particularly strong induction at 30 °C, reaching an increase of 205%. In contrast, XWX12 showed enhanced SOD activity mainly under higher Cd exposure, with increases ranging from 36.2% to 114% (Figure 4B). These results indicate that antioxidant defense systems were activated under combined stress, with XWX12 exhibiting a generally stronger enzymatic response, whereas YZX showed a comparatively moderate adjustment. Malondialdehyde (MDA) content, an indicator of lipid peroxidation, increased under both Cd and elevated temperature treatments. In YZX, MDA content increased significantly by 40.0% and 79.4% under 0 and 5 μmol L^−1^ Cd, respectively, but showed only a minor increase at 20 μmol L^−1^ Cd. In contrast, XWX12 exhibited significant increases in MDA content at all Cd concentrations (82.0%, 62.5%, and 46.6%), indicating more severe oxidative damage. Overall, under Cd stress, the temperature-induced increase in MDA accumulation was lower in YZX than in XWX12 (Figure 4C). Catalase (CAT) activity tended to decline with increasing Cd concentration but was generally enhanced by exposure to 30 °C. In YZX, CAT activity increased significantly by 42.0% and 22.8% under 0 and 5 μmol L^−1^ Cd, respectively, but showed no significant change at 20 μmol L^−1^ Cd. In XWX12, CAT activity increased slightly under all Cd treatments, although these changes were not statistically significant. Overall, temperature elevation significantly enhanced CAT activity in both cultivars, with a stronger induction observed in YZX (Figure 4D). Root activity declined with increasing Cd concentration in both cultivars. Under high Cd stress at the same temperature, YZX exhibited a more pronounced reduction in root activity. However, temperature elevation significantly increased root activity under all Cd treatments; YZX increased by 39.5% at 0 μmol L^−1^ Cd, and XWX12 increased by 45.2% and 52.6% at 5 and 20 μmol L^−1^ Cd, respectively (Figure 4E). Overall, under Cd-free conditions, temperature-induced stimulation of root activity was greater in YZX than in XWX12, whereas under identical Cd concentrations, root activity in YZX was more strongly affected by the combined effects of temperature and Cd stress.

2.4. Impact of Elevated Temperature of 30 °C on Chlorophyll Content and Photosynthetic Characteristics in Rice Varieties Under Cadmium Stress

Rice materials were grown in soils with cadmium concentrations of total Cd 1.52 mg/kg and available Cd 0.76 mg/kg. Elevated temperature (30 °C) modulated chlorophyll content and photosynthetic performance in rice seedlings under cadmium (Cd) stress, revealing varietal differences (Figure 5A–E). SPAD values increased in XWX12, rising by 7.8%, indicating partial mitigation of Cd-induced pigment loss (Figure 5A). Photosynthetic parameters also improved under warming, though patterns differed: YZX showed increases in net photosynthetic rate (Pn, 28.5%) (Figure 5C) and intercellular CO2 (Ci, 10.3%) (Figure 5D), whereas XWX12 exhibited larger gains in Tr (85.7%) (Figure 5B) and Gs (72.4%) (Figure 5E), with moderate Pn increase (31.6%) (Figure 5C) and minimal Ci change (Figure 5D). These results indicate that warming enhances carbon assimilation and gas exchange, with YZX prioritizing photosynthetic efficiency and XWX12 favoring stomatal and water-related adjustments. Overall, varietal responses under combined Cd and temperature stress differ, highlighting the importance of cultivar selection for rice production in Cd-contaminated and warming environments.

2.5. Transcriptomic Responses of Rice Cultivars to Combined Cadmium and Elevated Temperature Stress

The combined impact of cadmium (Cd) contamination and elevated temperature poses significant challenges for rice growth and ecological sustainability [22], yet the molecular mechanisms underlying cultivar-specific responses remain poorly understood. RNA-sequencing was conducted to characterize cultivar-specific transcriptomic responses of YZX and XWX12 to cadmium (Cd; 0 or 20 μmol L^−1^) under two temperature regimes (25 °C and 30 °C) for 5 and 10 days (Figure 6A–K and Figure S1). After stringent quality control, approximately 1.97 × 10^9^ clean reads (>85% of total reads) were retained, providing a robust foundation for downstream analyses (Table 1 and Table S1). Temperature treatments alone (25 °C vs. 30 °C) induced pronounced, time- and genotype-dependent transcriptional changes. After 5 days, XWX12 exhibited 2567 differentially expressed genes (DEGs; 1652 upregulated and 915 downregulated) (Figure 6A), whereas YZX showed a markedly stronger response with 3932 DEGs (2123 upregulated and 1809 downregulated) (Figure 6C). After 10 days, the total number of DEGs declined in both cultivars, with 1123 DEGs in XWX12 (697 upregulated, 426 downregulated) (Figure 6E) and 2716 in YZX (1699 upregulated, 1017 downregulated) (Figure 6G); however, upregulated genes remained predominant, indicating sustained transcriptional activation under prolonged temperature treatment. Cd treatment further reshaped transcriptomic profiles in a cultivar-specific manner under both temperature conditions. Under 20 μmol L^−1^ Cd for 5 days, XWX12 and YZX exhibited 2717 DEGs (1882 upregulated, 835 downregulated) (Figure 6B) and 2790 DEGs (1739 upregulated, 1051 downregulated) (Figure 6D), respectively. After 10 days of combined Cd and temperature treatment, DEG numbers decreased but remained substantial, particularly in YZX (2351 DEGs; 1796 upregulated) (Figure 6H), compared with XWX12 (960 DEGs; 689 upregulated) (Figure 6F). Comparative analyses revealed partial transcriptional acclimation over time. Venn diagram analysis identified 948 DEGs shared between temperature treatment and combined Cd–temperature treatment at 5 days (Figure 6I), whereas only 149 DEGs overlapped at 10 days (Figure 6J), indicating increasing divergence of transcriptional programs with prolonged treatment duration. Notably, 81 DEGs were consistently shared across both time points, representing a core set of genes potentially involved in sustained stress adaptation (Figure 6K). Overall, rice cultivars exhibited dynamic and genotype-dependent transcriptomic responses to temperature and Cd treatments, characterized by extensive early transcriptional reprogramming and sustained activation of stress-responsive genes during prolonged treatment.

2.6. GO and KEGG Enrichment Analyses Reveal Dynamic Molecular Responses to Cd and Elevated Temperature Stress

GO and KEGG enrichment analyses were performed on the identified differentially expressed genes (DEGs) (Figure S2 and Figure 7). At 5 days, a total of 948 DEGs were detected between temperature treatment and combined Cd–temperature treatment (Figure 6I). GO enrichment analysis revealed that these genes were predominantly associated with Biological Process terms related to cellular process, metabolic process, and response to stimulus. In the Molecular Function category, binding, catalytic activity, and transporter activity were significantly enriched, whereas Cellular Component terms were mainly represented by membrane, organelle, and extracellular region (Figure S2A). These results indicate that metabolic regulation, membrane-associated functions, and transmembrane transport are rapidly activated during the early response to combined temperature and Cd stress. By 10 days, the number of DEGs between temperature treatment and combined Cd–temperature treatment decreased to 149 (Figure 6J), yet GO enrichment patterns remained consistent across all three categories, suggesting the establishment of a more stable adaptive transcriptional program under prolonged stress conditions (Figure S2B). A combined analysis of DEGs from both time points further confirmed that metabolic processes, stress responsiveness, and transport-related functions constitute the core biological responses to temperature–Cd interactions(Figure S2C). KEGG pathway enrichment analysis provided additional insights into the underlying regulatory mechanisms. At 5 days, DEGs were significantly enriched in pathways associated with starch and sucrose metabolism, phenylpropanoid biosynthesis, and phenylalanine metabolism (Figure 7A). Notably, pathways directly implicated in stress mitigation, including ascorbate and aldarate metabolism and ABC transporters, were also significantly enriched, highlighting the importance of antioxidant defense and transmembrane transport in early responses to combined temperature and Cd toxicity (Figure 7A). At 10 days, the MAPK signaling pathway emerged as a central regulatory pathway under temperature–Cd co-stress (Figure 7B). Importantly, MAPK signaling remained significantly enriched when all DEGs from both time points were considered, underscoring its pivotal role in coordinating long-term signaling and adaptive responses to combined temperature and Cd stress in rice (Figure 7C).

2.7. Integrated Transcriptional Networks Linking Antioxidant Metabolism, Transport Systems, and Stress Signaling Under Combined Temperature and Cadmium Stress

Building on GO and KEGG enrichment results that highlighted ascorbate and aldarate metabolism, ABC transporters, and the MAPK signaling pathway as key pathways responding to combined temperature and cadmium (Cd) stress, we further integrated pathway-level and gene-level analyses to characterize coordinated transcriptional regulation in the two rice cultivars (Figure 8A–F). Genes associated with antioxidant metabolism were strongly induced in a temperature- and Cd-dependent manner, with elevated temperature (30 °C) markedly enhancing the expression of redox-related genes, including OsAPX, OsALDH3, OsGGP, glutathione S-transferases (OsGSTU5/6), thioredoxin (OsTRXh1), peroxidases (OsPRX2 and OsPrx41), and glutathione reductase (OsGR) (Figure 8A,E). This induction was particularly pronounced under combined temperature–Cd stress and was more sustained in XWX12 than in YZX, indicating enhanced antioxidant buffering capacity in XWX12. Consistent with enhanced detoxification demands, extensive activation of ABC transporter genes accompanied antioxidant responses. Multiple members of the ABCG and ABCB subfamilies (e.g., OsABCG13, OsABCG22, OsABCG31, OsABCB4, and OsABCG37) were preferentially induced under elevated temperature and Cd exposure, suggesting intensified transmembrane transport and detoxification of Cd or Cd-associated metabolites (Figure 8B). Compared with YZX, XWX12 displayed broader and more persistent induction of ABC transporters, supporting a cultivar-specific advantage in Cd handling efficiency. In parallel, genes directly involved in Cd uptake, sequestration, and intracellular transport, including OsNRAMP, OsHMA, OsMTP, OsPCR1, and OsCCX2, exhibited strong temperature-dependent regulation (Figure 8F). Elevated temperature promoted the expression of vacuolar sequestration-related genes such as OsMTP7 and OsMTP11 in XWX12, whereas YZX showed stronger induction of plasma membrane-associated transporters (OsNRAMP1/2 and OsHMA2), particularly under prolonged Cd exposure (Figure 8F). These contrasting expression patterns suggest distinct strategies of Cd partitioning between the two cultivars, with XWX12 favoring intracellular sequestration and YZX relying more on uptake and redistribution processes. Concurrently, core components of the MAPK signaling pathway, including OsMAPKKKα, OsMPK3, OsACTPK1, and calcium signaling regulators (OsCBL8 and OsCBL10), were consistently activated under temperature–Cd co-stress, especially at 30 °C and 10 d (Figure 8C). This sustained activation underscores the role of MAPK-mediated signaling as an integrative hub linking thermal perception, oxidative stress, and Cd-responsive transcriptional regulation. In addition, classical temperature-responsive genes, such as OsHsfA2, OsHSP70/90, OsbZIP23, OsBZR4, and OsNCED family members, were broadly induced, reflecting tight coupling between temperature signaling and Cd stress responses (Figure 8D). Collectively, these results demonstrate that elevated temperature amplifies Cd-induced transcriptional reprogramming through coordinated activation of antioxidant metabolism, ABC transporter–mediated detoxification, MAPK signaling, and Cd transport systems. The stronger and more sustained activation of these interconnected pathways in XWX12 compared with YZX suggests a mechanistic basis for cultivar-specific differences in tolerance to combined temperature and Cd stress.

2.8. Validation of RNA-Seq Data by Quantitative Real-Time PCR (qRT-PCR)

To validate the reliability of our RNA-seq data, we selected 15 differentially expressed genes (DEGs) for quantitative real-time PCR (qRT-PCR) analysis across all 32 experimental conditions. The expression patterns of the selected gene OsR498G0100010900.01 (OsTLP27) determined by qRT-PCR were highly consistent with the RNA-seq results, showing similar trends of up- or down-regulation under different cadmium and temperature treatments (Figure 9A). To further assess the overall correlation, we performed a linear regression analysis between the relative expression levels obtained from RNA-seq (FPKM) and qRT-PCR (2^−ΔΔCt^) for all 15 genes. The results revealed a strong positive correlation between the two datasets, with a Pearson correlation coefficient (r) of 0.873 (Figure 9B). The coefficient of determination (R^2^) was 0.763, indicating that the RNA-seq data could account for 76.3% of the variation observed in the qRT-PCR data. This strong and statistically significant correlation (p < 0.001) confirms the accuracy and reliability of our transcriptomic sequencing results.

3. Discussion

Against the backdrop of intensifying global climate warming and the increasing co-occurrence of heavy metal contamination in agricultural soils [23], growing attention has been directed toward the interactive effects of temperature and cadmium (Cd) on crop growth, metal accumulation, and stress tolerance mechanisms [24]. However, systematic investigations into how different rice genotypes respond to Cd stress under elevated temperature, particularly through integrated comparisons spanning phenotypic, physiological, and molecular levels, remain limited [23]. In this study, by combining morphological traits, physiological and biochemical measurements, and transcriptomic analyses, we comprehensively elucidate how warming reshapes rice growth performance, Cd uptake and partitioning, and the underlying molecular regulatory networks, demonstrating that elevated temperature acts as a catalyst that amplifies genotype-dependent adaptive strategies.

Under the combined pressures of global climate warming and soil heavy metal contamination, understanding how interacting environmental factors shape crop growth has become central to sustainable agriculture [25,26]. In this study, moderate warming to 30 °C consistently promoted early seedling growth in both rice cultivars, as evidenced by increased plant height, root length, and biomass, in agreement with previous reports showing that near-optimal temperatures enhance early rice development [27] (Figure 1). Moreover, within the Cd concentration range of 0–5 μmol L^−1^, exposure to 30 °C significantly alleviated the inhibitory effects of Cd on plant height, root length, and fresh weight, although the magnitude of this compensatory response varied markedly between genotypes (Figure 1). YZX exhibited a pronounced increase in plant height under low Cd conditions, whereas XWX12 primarily adapted by maintaining a longer root system (Figure 1). This genotype-specific strategy is consistent with observations reported that Cd-tolerant rice genotypes commonly prioritize root elongation to sustain water and nutrient acquisition [27]. Crucially, our results demonstrate that elevated temperature exacerbates genotype-dependent growth differences under Cd stress. Unlike previous studies conducted at constant temperatures [27], we show that warming creates a specific selective pressure that magnifies the trade-off between biomass accumulation and stress avoidance.

As a key driver of heavy metal bioavailability, climate warming can profoundly reshape elemental uptake [28], translocation, and partitioning within plants [29], thereby determining genotype-specific patterns of metal accumulation [30]. We found that warming to 30 °C markedly increased root Cd concentrations in both cultivars, with YZX consistently exhibiting higher Cd accumulation and translocation coefficients across treatments; these differences were further amplified under combined high Cd and elevated temperature (Figure 2 and Figure 3). This pattern aligns with the strong upregulation of plasma membrane–localized Cd transporters [30,31], including OsNRAMP [31,32] and OsHMA genes [33,34], in YZX, indicating a strategy biased toward enhanced uptake and long-distance transport (Figure 8F). In contrast, XWX12 showed stronger induction of vacuolar sequestration–related genes such as OsMTP7 and OsMTP11 under warming [35,36,37], favoring intracellular immobilization of Cd and restricting its root-to-shoot movement [1] (Figure 8F). Similar contrasts between Cd-tolerant and Cd-sensitive rice genotypes have been reported previously [38,39], where tolerant genotypes limit root–shoot translocation while sensitive genotypes exhibit a “high-accumulation–high-sensitivity” profile comparable to YZX; however, those studies were conducted under fluctuating day–night temperatures and did not resolve the specific role of constant warming. By explicitly incorporating sustained temperature elevation, our results demonstrate that warming not only enhances Cd influx into roots but also magnifies genotypic differences in translocation efficiency, highlighting temperature sensitivity as a critical, yet underappreciated, criterion for breeding low-Cd rice under future climate scenarios. By explicitly incorporating sustained warming, we establish that temperature sensitivity serves as an amplifier of translocation efficiency, differentiating “avoider” (XWX12) and “accumulator” (YZX) phenotypes more sharply than Cd stress alone.

Our data further indicates that the growth advantage established at the seedling stage persists through the reproductive stage (Figure 3). Consistent with our findings, pot experiments under Free Air Temperature Increase (FATI) conditions showed that warming significantly increased Cd concentrations in rice grains and enhanced Cd transfer from roots to shoots [40,41]. Similarly, it was reported that elevated temperatures disproportionately favored biomass accumulation in high-Cd-accumulating genotypes relative to low-accumulating ones. These results collectively indicate that genotype × temperature interactions influence growth and Cd dynamics across both controlled and field environments [42], suggesting that genotype × temperature interactions are stable across the entire growth period. Root Cd concentration in YZX increased by 60% under 30 °C, exceeding the 36% increase in XWX12 (Figure 2 and Figure 3). This pattern is consistent with previous studies using the same soil temperature gradient (25–30 °C), where root Cd accumulation responded more strongly to warming in high-accumulating cultivars than in low-accumulating ones [42,43]. This further supports the conclusion that elevated temperature amplifies genotype-specific differences in Cd uptake, likely via enhanced root transpiration and mass flow.

Cadmium stress disrupts cellular homeostasis through ROS overaccumulation, making antioxidant systems central to Cd tolerance [44,45]. In this study, POD and SOD activities were markedly induced under Cd and elevated temperature (Figure 4A,B), with XWX12 showing stronger and more sustained responses, consistent with transcriptomic evidence of early enrichment in ascorbate–glutathione metabolism and ABC transporter pathways (Figure 7A). Although POD activity in XWX12 plateaued under high Cd stress (20 μmol L^−1^), the significant upregulation of SOD (Figure 4B) suggests a compensatory regulation within the antioxidant network to mitigate the combined oxidative stress of Cd and warming. This likely underlies XWX12’s reduced Cd accumulation in shoots via enhanced detoxification and compartmentalization. MDA levels increased in both cultivars, but temperature-induced lipid peroxidation was lower in YZX, indicating milder oxidative damage (Figure 4C). CAT activity was strongly enhanced by 30 °C, especially in YZX (42%), in line with heat-induced OsCATB upregulation, suggesting H_2_O_2_-mediated transcriptional activation [46] (Figure 4D). Root activity exhibited genotype- and Cd-dependent responses: XWX12 increased root activity under high Cd with warming, possibly due to active cytokinin synthesis, whereas YZX showed a pronounced decline, implying that root activity changes mainly reflect membrane permeability rather than metabolic capacity (Figure 4E). Collectively, these results highlight early antioxidant–transport coordination and genotype-specific root responses as key determinants of rice tolerance to combined Cd and temperature stress.

Exposure to 30 °C improved SPAD values and net photosynthetic rate (Pn) in both cultivars, consistent with previous reports that moderate warming stabilizes the photosynthetic apparatus under Cd stress [47] (Figure 5A,C). Notably, the two genotypes adopted contrasting resource-allocation strategies. YZX primarily enhanced carbon assimilation capacity, as reflected by pronounced increases in Pn and stomatal conductance (Gs), conforming to a “high photosynthesis–high growth” strategy (Figure 5C,E). In contrast, XWX12 exhibited a disproportionate increase in transpiration rate (Tr; +86%), suggesting that transpiration-driven mass flow may promote Cd dilution in leaf tissues (Figure 5B). This response provides experimental support, for the first time in rice, for the “transpiration-driven Cd dilution” hypothesis proposed. Importantly, these findings identify a high Tr-to-Pn ratio as a practical physiological indicator for selecting low-Cd-accumulating cultivars under warming conditions.

GO and KEGG enrichment analyses revealed a clear temporal shift in the molecular responses to combined temperature and Cd stress. At the early stage (5 d), shared differentially expressed genes (DEGs) were predominantly enriched in ABC transporters and phenylpropanoid metabolism (Figure 7A), whereas sustained activation of the MAPK signaling pathway became prominent by 10 d (Figure 7B), with consistently stronger induction in XWX12 than in YZX. A similar “MAPK–ABC” synergistic regulatory pattern was reported in OsNAC5-mutant lines [48], where suppression of OsNAC5 attenuated MAPK cascade signaling [48], accompanied by reduced expression of ABCG family transporters and increased Cd accumulation [49]. Collectively, this study establishes a conceptual framework for understanding crop adaptation to heavy metal stress under climate warming and offers molecular targets of practical relevance for breeding Cd-tolerant, low-accumulation rice cultivars and improving risk management in contaminated agroecosystems. In summary, this study provides a mechanistic framework for crop adaptation, identifying the MAPK-ABC module and physiological trade-offs as critical targets for breeding climate-resilient, low-Cd rice.

4. Materials and Methods

4.1. Plant Materials and Growth Conditions

Two late-season rice (Oryza sativa L.) varieties, YZX (high-Cd-accumulating, temperature-sensitive) and XWX12 (low-Cd-accumulating, temperature-tolerant), were provided by Hunan Kehui Seed Industry Co., Ltd. (Changsha, China). Experiments were conducted at the seedling and maturity stages. For seedlings, plump seeds were surface-sterilized with 75% ethanol (3 min), rinsed 4–5 times, and soaked in distilled water at 37 °C for one day. Uniformly germinated seeds were transferred to hydroponic containers, cultured in water for three days, then in Kimura B nutrient solution, refreshed every three days. Growth conditions were 30,000 lx, 12 h light/12 h dark at 30 °C, and 70% relative humidity. At the two-leaf-one-heart stage, seedlings were treated with temperatures of 25 °C or 30 °C and Cd concentrations of 0, 5, or 20 µmol/L for 10 days. For the maturity stage, germinated seeds were sown in nutrient soil and transplanted at the four-leaf-one-heart stage into Cd-contaminated soil from Huayuan Village, Liuyang City (Quaternary red soil; pH 5.53; organic matter 21.413 g/kg; total Cd 1.52 mg/kg; available Cd 0.76 mg/kg). Transplanted seedlings were grown either in a plastic greenhouse (30 °C) or outdoors, with the temperature difference recorded at maturity.

4.2. Phenotypic and Biomass Measurements

At the conclusion of the seedling stage treatments, ten seedlings were randomly selected from each treatment group for phenotypic measurements. Plant height was measured from the stem base to the tip of the longest leaf, and root length was measured as the length of the longest root, both using a steel ruler. For biomass determination, randomly selected seedlings were gently blotted with absorbent paper to remove excess water from the roots. The plants were then separated into shoots and roots, and their fresh weights were immediately measured using a one-ten-thousandth analytical balance. Subsequently, the samples were deactivated in an oven at 120 °C for 30 min and then dried at 70 °C to a constant weight to determine the total dry matter.

4.3. Determination of Cadmium Content

Plant samples were prepared for Cd analysis after treatments. Seedling roots were washed in 20 mmol/L EDTA-Na_2_ (20 min) to remove surface Cd, then rinsed with ultrapure water. Plants were separated into root, stem, and leaf, oven-dried at 70 °C to constant weight after deactivation at 120 °C for 1 h, and ground into fine powder. About 0.25 g of powder (±0.001 g) was digested in 10 mL nitric acid: perchloric acid (4:1 v/v) overnight, then on a 230 °C block with swirling every 30 min until digestion was completed. The digestate was cooled, transferred, and diluted to 50 mL. After sedimentation, the supernatant was collected and further diluted (seedling roots 2000×, stems 500×, maturity-stage roots 10×). Cd concentration was measured by graphite furnace atomic absorption spectrophotometry (GFAAS) at 228.8 nm with Zeeman background correction. Cd content was calculated from instrument readings, sample weight, final volume, and dilution factor. Cd translocation ratio was the ratio of Cd in the stem or leaf to the root.

4.4. Measurement of Physiological and Biochemical Indicators

For the determination of antioxidant enzyme activities, frozen plant samples (−80 °C) were ground to a fine powder in liquid nitrogen using a pre-chilled mortar and pestle. For each of three biological replicates, 0.5 g of powdered tissue was suspended in 5 mL of pre-chilled phosphate buffer (pH 7.8) on ice. The homogenate was centrifuged at 4200 rpm for 20 min at 4 °C. The resulting supernatant was collected and centrifuged again at 13,000 rpm for 10 min at 4 °C to obtain the final crude enzyme extract, which was stored at 4 °C for immediate analysis. Peroxidase (POD) activity was measured using the guaiacol oxidation method. The reaction mixture contained 3 mL of a freshly prepared POD reaction solution (0.05% guaiacol and 0.003% H_2_O_2_ in pH 7.0 phosphate buffer) and 0.1 mL of crude enzyme extract. The reaction was initiated by adding the enzyme extract and incubating at 37 °C for 15 min, then terminated by adding 2 mL of 20% trichloroacetic acid. The absorbance of the resulting colored product was measured at 470 nm against a blank control. POD activity was expressed as U/(g·min) based on the change in absorbance, total extract volume, sample fresh weight, enzyme volume used, and reaction time.

Superoxide dismutase (SOD) activity was determined by measuring its ability to inhibit the photochemical reduction of nitro-blue tetrazolium (NBT). The 3.3 mL reaction mixture contained 1.5 mL of pH 7.8 phosphate buffer, 0.3 mL of 65 mmol/L methionine, 0.3 mL of 500 µmol/L NBT, 0.3 mL of 100 µmol/L EDTA-Na_2_, 0.3 mL of 200 µmol/L riboflavin, 0.1 mL of enzyme extract, and 0.5 mL of distilled water. The reaction was illuminated at 4000 lx for 25 min. A dark control was wrapped in foil, and a light control contained buffer instead of enzyme extract. The absorbance was measured at 560 nm, with the dark control used as a blank. SOD activity, defined as the amount of enzyme required to cause 50% inhibition of the NBT reduction rate, was expressed as U/g fresh weight. Malondialdehyde (MDA) content, an indicator of lipid peroxidation, was measured using the thiobarbituric acid (TBA) reaction. 0.5 g of fresh tissue was homogenized in 5 mL of distilled water, followed by the addition of 5 mL of 0.5% TBA in 20% trichloroacetic acid. The mixture was heated in a boiling water bath for 10 min, cooled, and centrifuged. The absorbance of the supernatant was measured at 450 nm, 532 nm, and 600 nm. MDA content was calculated using a corrective formula based on the absorbance values and expressed as µmol/g fresh weight.

4.5. Photosynthetic Parameter Measurement

Relative chlorophyll content was non-destructively measured on the second-to-last fully expanded leaf of the main stem using a portable SPAD-502PLUS chlorophyll meter (Konica Minolta, Tokyo, Japan). Three leaves were selected per pot, and three readings were taken per leaf, with the average value representing the SPAD reading for that treatment. Gas exchange parameters were measured at the maturity stage between 9:00 and 11:00 a.m. on clear days using a LI-6800 portable photosynthesis system (LI-COR, Lincoln, NE, USA). Measurements were taken on the second-to-last functional leaf of five representative plants per treatment. The conditions in the leaf chamber were controlled at a CO_2_ concentration of 400 µmol/mol, a leaf temperature of 30 °C, a photosynthetic photon flux density (PPFD) of 800 µmol m^−2^s^−1^, and a flow rate of 500 µmol/s. The net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO_2_ concentration (Ci), and transpiration rate (Tr) were recorded.

4.6. Transcriptome Sequencing and Analysis

For transcriptome analysis, root samples were collected from seedlings of both varieties at the two-leaf-one-heart stage after 5 and 10 days of treatment with 0 µmol/L and 20 µmol/L Cd under both control (25 °C) and elevated (30 °C) temperatures. Three biological replicates were prepared for each condition. Roots were washed in 20 mM EDTA-Na_2_ and rinsed with deionized water, then immediately flash-frozen in liquid nitrogen and stored at −80 °C. Total RNA extraction, library construction, and sequencing on the Illumina HiSeq 4000 platform were performed by Huazhi Biotechnology Co., Ltd. (Changsha, China). After quality control filtering of raw reads, clean reads were aligned to the rice reference genome. Gene expression levels were quantified, and differentially expressed genes (DEGs) were identified using a threshold of an absolute log_2_ fold change > 1 and a p-value < 0.05. Functional annotation and enrichment analyses of the DEGs were conducted using Gene Ontology (GO) to categorize genes into biological process, cellular component, and molecular function domains, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) to identify enriched metabolic and signaling pathways.

4.7. Extraction of RNA from Rice Plants and Quantitative Real-Time PCR (qRT-PCR)

Take an appropriate amount of fresh rice leaf tissue and immediately place it in a pre-cooled mortar, adding liquid nitrogen for rapid grinding. Carefully prevent the complete evaporation of liquid nitrogen to avoid moisture in the powder. Transfer the thoroughly ground powder into a pre-cooled 2 mL RNA enzyme-free centrifuge tube, add 1 mL of Trizol lysis buffer, and mix vigorously on a vortex shaker. Immediately place the mixture on ice and let it stand for 10 min. Then centrifuge at 4 °C for 5–8 min at 12,000 rpm. Carefully aspirate the supernatant and transfer it to a new sterilized 1.5 mL centrifuge tube. Add 500 μL of chloroform, vortex to emulsify, and mix thoroughly. Let the mixture stand at 4 °C for 10 min. Centrifuge again at 4 °C for 10 min at 12,000 rpm. Aspirate 400 μL of the clear, colorless aqueous phase into a 1.5 mL RNA enzyme-free centrifuge tube, ensuring no white flocculent material is collected. If white flocculent is observed, re-centrifuge. Add 400 μL of pre-cooled isopropanol (equal volume) and mix thoroughly. Let the mixture stand at 4 °C for 10–30 min. Centrifuge at 4 °C for 10 min at 12,000 rpm. Discard the supernatant and retain the white precipitate. In the tube containing the white precipitate, add 1 mL of RNase-free ddH2O diluted with 75% ethanol, and centrifuge at 12,000 rpm for 5 min. After two rounds of washing, discard the supernatant, open the lid, and let it stand at room temperature for 5 min to dry, then add 50 μL of RNase-free ddH2O and mix thoroughly. Store in an ultra-low temperature freezer at −80 °C. Using the Vazyme Reverse Transcriptase Kit, extract RNA from the freezer, thaw it on ice, and measure the RNA concentration and purity precisely with a Nanodrop. Calculate the RNA content based on the template concentration, then add it to the reverse transcription-PCR system. Mix thoroughly and incubate in a PCR machine at 42 °C for 2 min to remove genomic DNA. After the first reaction step, add 5× HiScript II qPT SuperMix II to convert RNA to cDNA. Mix thoroughly and incubate in a PCR machine at 50 °C for 15 min, followed by 85 °C for 5 s. For qPCR, use Actin as the internal reference gene. Data analysis employs the CT (2^−ΔΔCt^) method to calculate gene expression levels, with each sample replicated three times.

4.8. Data Analysis

Experimental data were initially processed using Microsoft Excel. Statistical analyses, including analysis of variance (ANOVA) and Least Significant Difference (LSD) multiple comparison tests, were performed using DPS 7.55 software. All charts and graphs were generated using GraphPad Prism (version 8.01) software.

5. Conclusions

This study demonstrates that elevated temperature and cadmium (Cd) stress interactively influence rice growth, Cd accumulation, and stress physiology in a genotype-dependent manner.

(1)Yuzhenxiang (YZX) exhibited a “high-accumulation–high-translocation” strategy, where warming amplified Cd redistribution to shoots under warming, Xiangwanxian 12 (XWX12) displayed a “root-retention–low-translocation” strategy, prioritizing root elongation and restricting root-to-shoot Cd transfer via enhanced intracellular Cd sequestration.(2)Regarding antioxidant defense, both cultivars activated enzymatic systems, but with distinct regulatory patterns: XWX12 exhibited a sustained upregulation of SOD and broader induction of ABC transporter genes to mitigate oxidative damage, whereas YZX primarily showed significant induction of POD and CAT activities.(3)Transcriptomic analyses further revealed that the coordinated activation of antioxidant metabolism, the MAPK signaling pathway, and ABC transporters constitutes a key molecular mechanism underlying cultivar-specific adaptation.(4)Overall, these results indicate that rice cultivars deploy distinct physiological and molecular strategies to cope with simultaneous Cd and elevated temperature stress, providing valuable insights for cultivar selection and breeding programs aimed at improving tolerance and food safety under warming and Cd-contaminated environments.