Genotypic Difference of Germination and Early Seedling Growth in Response to Cd Stress and its Relation to Cd Accumulation

A greenhouse experiment was conducted using twelve Japonica rice genotypes differing in grain Cd concentration, to study the genotypic difference in germination and early seedling growth in response to Cd stress and its relation to Cd accumulation. The results showed that under low Cd level (0.5 mM), germination was significantly enhanced for most rice genotypes, but higher Cd levels (2.5 and 10.0 mM) inhibited germination dramatically. When exposed to 10.0 mM Cd, a few genotypes could still germinate, but the seedlings of all genotypes would die in early stage. Shoot height and dry weight, and root length and dry weight of seedling were significantly inhibited at 0.5 and 2.5 mM Cd levels, irrespectively of genotypes, and the inhibition became more severe under higher Cd level. There was a significant genotypic difference in response of germination, early seedling growth and shoot Cd concentration to Cd stress. The difference was the largest for germination, followed by root growth, the least for shoot growth. A cluster analysis of tolerance indices, including germination rate, shoot height and weight, root length and weight of the plants exposed to 2.5 mM Cd level showed that 12 rice genotypes may be grouped into three clusters, i.e. high-, intermediate- and weak Cd-tolerance. No significant correlation was found between grain Cd concentration and Cd-tolerance in terms of germination and seedling growth, and shoot Cd concentrations, suggesting that it is possible to develop rice cultivars with high Cd-tolerance and low grain Cd accumulation.     

Responses of Components of Antioxidant System in Moongbean Genotypes to Cadmium Stress

Four genotypes (Pusa 9531, Pusa 9072, Pusa Vishal, PS‐16) of moongbean [Vigna radiata (L.) Wilczek] grown in earthen pots were treated with cadmium at 0, 25, 50, and 100 mg kg^?1 soil. Cadmium tolerance (CdT), the ability of a plant to maintain growth at high levels of cadmium (Cd), was calculated as the ratio of dry‐matter production in the untreated and the Cd‐treated soils. The moongbean genotypes showed a differential response to Cd concentrations; Pusa 9531 was identified as Cd tolerant, whereas PS 16 was Cd susceptible. To find out the physiological basis of these differences, we investigated the possible role of antioxidant (enzymatic and nonenzymatic) defense systems. Activities of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6), ascorbate peroxidase (EC 1.11.1.11), and glutathione reductase (EC 1.6.4.2) and the amounts of ascorbate and glutathione were monitored in the Cd‐tolerant and Cd‐sensitive moongbean genotypes. The results revealed the presence of a strong antioxidant defense system in the Cd‐tolerant genotype (Pusa 9531) for providing adequate protection against oxidative stress caused by Cd.     

Arbuscular mycorrhizal fungi inoculation and exogenous indole-3-acetic acid application induce antioxidant defense response to alleviate cadmium toxicity in Broussonetia papyrifera

Cadmium (Cd) contamination in soil poses a huge threat to plants even at low concentrations; Broussonetia papyrifera has great potential in remediation of soil heavy metal contamination. However, whether exogenous indole-3-acetic acid (IAA) application and arbuscular mycorrhizal fungi (AMF) have synergistic effects on Cd tolerance of B. papyrifera remains unclear. To investigate the effects of AMF inoculation and IAA application on the tolerance of B. papyrifera to Cd stress, two experiments were conducted: the first to investigate the effect of AMF (Rhizophagus irregularis) inoculation on the tolerance of B. papyrifera to Cd stress and the second to investigate the combined effects of AMF inoculation and IAA application on the tolerance of B. papyrifera to Cd stress. Parameters including endogenous hormone concentration, antioxidant defense response, malondialdehyde (MDA) content, and gene expression related to antioxidant enzyme system and hormone were measured. The results indicated that AMF alleviated Cd toxicity of B. papyrifera by reducing MDA content and improving antioxidant enzyme activities and Cd absorption capacity. Furthermore, the combination of AMF inoculation and IAA application had a synergetic effect on the tolerance of B. papyrifera to Cd stress through upregulating BpAUX1 and BpAUX2, which might contribute to root growth and root xylem synthesis, and by upregulating BpSOD2 and BpPOD34 to enhance the antioxidant enzyme system. This work provides a new insight into the application of IAA in the remediation of soil Cd pollution by mycorrhizal plants.     

硅对Cd胁迫下黄瓜苗期光合及抗氧化酶系统的影响

为了解石灰性土壤硅对Cd胁迫下黄瓜苗期叶片的光合特性和抗氧化酶系统及对实际土壤Cd毒害植物的防治,研究了硅介入下Cd污染土壤中黄瓜苗期的叶片净光合速率(P_n)、气孔导度(G_s)、蒸腾速率(T_r)、胞间CO_2(C_i)等光合作用指标,叶绿素含量,丙二醛(MDA)、超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)等抗氧化酶系统等相关参数。结果表明:与空白相比较,单施Cd处理下,P_n、G_s、T_r和叶绿素含量下降,Ci提高。硅介入后,能明显提高P_n、G_s、T_r和叶绿素总量,降低C_i含量。当C_d含量为5mg/kg时,硅施加量100~200mg/kg情况下,与空白相比,P_n、G_s、T_r和叶绿素上升,C_i下降,但100mg/kg与200mg/kg的硅处理差异不明显,300mg/kg的硅添加量效果最好。对于抗氧化酶系统,与空白相比较,土壤Cd含量为3~5mg/kg时,3类抗氧化酶的活性均受到显著抑制,200~300mg/kg硅的引入可明显提高SOD、POD、CAT活性,降低MDA值。可见,硅的引入可明显改善Cd胁迫下黄瓜叶片的光合特性和抗氧化酶系统。施用少量硅肥在石灰性土壤中对缓解植物Cd毒害具有一定的应用前景。     

A study on the effect of cadmium on the antioxidative defense system and alteration in different functional groups in castor bean and Indian mustard

The present study was planned to delineate the role of antioxidants and different functional groups of Ricinus communis and Brassica juncea in the tolerance mechanisms toward cadmium (Cd) for phytoremediation. Application of Cd caused a reduction in dry biomass of 53.84% and 26.58% in root and 45.33% and 33.84% in shoots of B. juncea and R. communis, respectively. Antioxidant enzymes, namely superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase, glutathione reductase and glutathione-S-transferase, and metabolites (proline) increased in both the species due to Cd exposure. The metal caused substantial changes in the functional groups present in the roots and leaves of the plants. A number of new peaks appeared in the Cd-treated plants, which indicate the production of the compounds responsible for the metal tolerance of these plants. R. communis has been found to possess a good antioxidant defense system against Cd stress and may be used for the phytoremediation of metal-contaminated soils in place of edible crops, which enhance the risk of contaminating the food chain. It has been observed that R. communis accumulated 213.39 and 335.68 mg Cd in roots and shoots, respectively, whereas B. juncea accumulated 28.19 and 310.15 mg Cd in the roots and shoots, respectively.     

Response of leguminosae to cadmium exposure

Bush bean and pea plants grown in a sandy substrate and treated daily with nutrient solutions containing either 50 and 125 pM cadmium (Cd), added as cadmium nitrate [Cd(NO₃)2], were analyzed for dry matter production, total Cd content, and extractable Cd. Cadmium depressed dry matter production of both plant species. Bush bean plants accumulated larger amounts of Cd in tissues and displayed lower Cd tolerance than pea plants. The high accumulation of Cd in roots of bush bean does not seem to prevent Cd translocation. Pea plants show a higher exclusion capacity at the root level, suggesting that membrane selectivity rather than apoplastic compartmentation may act as a defence mechanism against Cd toxicity. Gel‐permeation chromatography and voltammetric analyses showed that part of water‐soluble Cd extracted from tissues of pea and bush bean was as free metal ion (Cd²⁺). In addition, Cd into the nutrient solution induced progressively the synthesis of water‐soluble proteins at low molecular weigth in tissues of both plant specie. In root extracts of pea and bush bean, Cd was also associated with “like‐protein”; fraction with apparent molecular weight >30 KDa.     

Stem-specific cadmium accumulation in cadmium-tolerant Polygonum thunbergii

Polygonum thunbergii was selected among wild plants as a cadmium (Cd)-tolerant plant because of the high accumulation of Cd in the stems. However, the concentration of Cd in the leaves was low, which suggested the existence of an inhibitory mechanism of Cd translocation from stems to leaves. Observation of Cd distribution in stem-petiole-lamina in P. thunbergii plants that had absorbed Cd, using SEM-EPMA, revealed that Cd accumulation occurred mainly in the nodes, junctions of petioles, and vessels in stems, with minimal translocation in petioles. A detection method for tissue distribution of Cd was developed. Resin-fixed tissue tips were stained with a fluorescent probe BTC-5N and then sites of Cd location were detected with a fluorescence microscope. It was found that Cd in the stem tissues of P. thunbergii was distributed at specific sites of the cell walls of vessels and surrounding cells. The results were consistent with the findings of the investigation of Cd distribution in stem cells obtained by the cell fractionation method, which showed that most of Cd was present in the cell wall and cytoplasm. These results suggest that P. thunbergii accumulates Cd in specific cell walls of the stems and inhibits Cd-translocation to the leaves.     

Vanadium induced manganese toxicity in bush bean plants grown in solution culture

Seedlings of two bush bean cultivars (Phaseolus vulqaris L. cvs. Mn‐sensitive ‘Wonder Crop 2’ and Mn‐tolerant ‘Green Lord') were grown for 14 days in full strength Hoagland No. 2 nutrient solution containing 0.05 ‐ 2 mg L^‐1 of vanadium (V) as ammonium vanadate.

Increasing V concentration in the solution decreased total dry weight of both cultivars. Plant tops were stunted and leaf color became dark green at 1 ‐ 2 mg L^‐1 V, especially in ‘Green Lord’. Veinal necrosis similar to that of Mn toxicity was observed in the primary leaves of ‘Wonder Crop 2’ at 0.2 mg L^‐1 V or above, but not in those of ‘Green Lord’.

The V concentrations in the roots increased exponentially with increasing V concentration in the solution; however, V concentrations in the leaves and stems were not affected. The Mn concentrations in the primary leaves increased under the higher V treatment in ‘Wonder Crop 2'; but not in ‘Green Lord’. In contrast, Fe concentration in the leaves of ‘Wonder Crop 2’ decreased markedly with increasing V concentration in the solution. Enhanced Mn uptake and greater reduction of Fe uptake by ‘Wonder Crop 2’ may explain the incidence of V‐induced Mn toxicity.     

重金属镉胁迫下入侵植物黄顶菊表观遗传变异特征

为探究入侵植物黄顶菊在重金属镉(Cd)胁迫下耐受性获得的表观遗传机制,本研究通过网室盆栽试验模拟不同浓度Cd污染生境,采用甲基化敏感扩增多态性(MSAP)技术对不同Cd浓度胁迫[0(CK)、2(Cd-1)、4(Cd-2)和8(Cd-3) mg·kg^-1]处理植物叶片基因组DNA甲基化变异特征进行分析。结果表明,15对引物共扩增出726条甲基化条带,且引物多态性百分比为84.75%;随着Cd胁迫浓度的升高,黄顶菊叶片全甲基化和整体甲基化发生比例呈逐渐增加的趋势,Cd-1、Cd-2和Cd-3的全甲基化发生比例分别为CK的1.51、1.95和2.11倍,整体甲基化发生比例分别较CK升高了39.28、53.30和63.97个百分点;不同处理下叶片甲基化状态变化分析结果表明,Cd胁迫下黄顶菊基因组DNA重新甲基化和去甲基化2种甲基化模式均有发生,但以重新甲基化类型为主要变化模式;Cd胁迫下植物表型可塑性与表观遗传相关性分析结果表明,DNA全甲基化和整体甲基化水平与黄顶菊生长指标及地上部耐受性指数的表型可塑性呈显著负相关,与抗氧化酶活性、各组织Cd含量及富集与转移系数呈显著正相关。本研究结果从表观遗传学方向为入侵植物黄顶菊的防控提供了新思路。     

Interaction of Salinity and Cadmium Stresses on Antioxidant Enzymes,Sodium, and Cadmium Accumulation in Four Barley Genotypes

A hydroponic experiment was carried out utilizing four barley genotypes with differing salt tolerances to investigate the influence of salinity (NaCl) and cadmium (Cd) on the activities of antioxidant enzymes, sodium (Na) and Cd concentrations, and accumulation in plants. The activities of the two antioxidant enzymes, superoxide dismutase (SOD) and peroxidase (POD), were significantly increased when plants were exposed to both Na and Cd stresses for all genotypes, relative to the control. The increased enzymatic activity was more predominant with a prolonged time of stress exposure. The combined stress (NaCl+Cd) led to a further increase in POD activity, but had little effect on SOD activity. Two salt-tolerant genotypes, ‘Gebeina’ and ‘Zhou 1,’ showed a more rapid increase of POD and SOD activities than the two salt-sensitive genotypes, ‘Newgoutei’ and ‘Quzhou’ in response to the combined stress treatment. Additions of NaCl to the Cd-containing medium caused a significant reduction in both Cd concentration and accumulation. The extent of the reduction in Cd concentration was also dependent on genotypes. The salt-tolerant genotypes had lower Na concentrations than sensitive genotypes, and the effect of Cd stress on Na concentration and accumulation varied with genotypes. It may be concluded that a significant interaction exists between Na and Cd in their influence on antioxidant enzyme activity and the accumulation of each element in the plant.     

Role of selenium in mitigation of cadmium toxicity in pepper grown in hydroponic condition

Selenium (Se) is an essential element for humans but is not considered as essential for plants. However, its beneficial role in improving plant growth and stress tolerances is well established. In order to study the role of Se in cadmium (Cd) toxicity in pepper (Capsicum frutescens cv. Suryankhi Cluster), this experiment was carried out in greenhouse conditions. Treatments comprised Cd [0, 0.25, and 0.5 mM cadmium chloride (CdCl₂)] and Se [0, 3, and 7 µM sodium selenite (Na₂SeO₃)] with three replications. The result showed that Cd decreased chlorophyll a, chlorophyll b, and carotenoids, whereas Se supplementation diminished Cd toxicity on photosynthetic pigment. Selenium at 7 µM significantly increased the leaf area in the plants grown at 0.25 mM Cd. The application of Se at 3 µM with 0.25 mM Cd and Se at 3 µM and Se at 7 µM with 0.5 mM Cd increased the activity of catalase (CAT). Selenium at 7 µM decreased the proline content of pepper leaves exposed to Cd at 0.5 mM (30%). Selenium significantly enhanced the antioxidant activity of leaves, which was diminished by Cd toxicity. In general, Se has a beneficial effect on plant growth and is an antioxidant enzyme of pepper cv. Suryankhi Cluster under Cd stress and non-stress conditions.     

Differential tolerance of bush bean cultivars to excess manganese in solution and sand culture

Nineteen bush bean cultivars were screened for tolerance to excess Mn in nutrient solution and sand culture experiments. Seven‐day‐old seedlings were treated with full strength Hoagland No. 2 nutrient solution containing different Mn concentrations for 12 days in the greenhouse.

Cultivars showing the greatest sensitivity to Mn toxicity were ‘Wonder Crop 1’ and ‘Wonder Crop 2'; those showing the greatest tolerance were ‘Green Lord’, ‘Red Kidney’ and ‘Edogawa Black Seeded’.

Leaf Mn concentration of plants grown in sand culture was higher than that for plants grown in solution culture. The lowest leaf Mn concentration at which Mn toxicity symptoms developed, was higher in tolerant than in sensitive cultivars. The Fe/Mn ratio in the leaves at which Mn toxicity symptoms developed, was higher in the sensitive cultivars than in the tolerant ones.

We concluded that Mn tolerance in certain bush bean cultivars is due to a greater ability to tolerate a high level of Mn accumulation in the leaves.     

Cadmium Effects in Sunflower: Membrane Permeability and Changes in Catalase and Peroxidase Activity in Leaves and Calluses

Mechanisms preventing oxidative burst in cells exposed to high metal concentrations are crucial for cell survival. In this report, sunflower (Helianthus annuus L.) was used as a model species to investigate: (a) how cadmium (Cd) affects plant defense pathways (in particular, those involved in preventing oxidative stress), and (b) whether antioxidative enzymes of plants and of in vitro cell culture (calluses) have similar responses to Cd exposure. For this experiment, plants (grown hydroponically on Long Ashton medium) and calluses (grown on Murashige and Skoog medium) were exposed for three weeks to different Cd concentrations (0, 5, 50, and 500 μ M). Solute and electrolyte leakage increased significantly with the increase in Cd concentration in the external medium and in exposed leaves, and less in exposed calluses. Cadmium exposure also reduced significantly soluble protein contents in both leaves and calluses. The activities of catalase and peroxidase decreased significantly in 50 and 500 μ M-exposed leaves compared with the control, but increased in 5 μ M-exposed calluses, decreasing in the calluses exposed 500 μ M Cd. The decrease in antioxidative enzymes activities is congruent with the decrease in membrane integrity and suggests that calluses develop antioxidant mechanisms that respond better to Cd stress than do leaves. On average, 100% of plants exposed for 21 d to 500 μ M and 95% of plants exposed for 45 d to 50 μ M Cd were dead. After 45 d, only 53% of 50 μ M-exposed calluses had died. These surviving calluses were maintained on 50 μ M for six months. Six-month-old exposed calluses had higher activities of peroxidase and catalase when compared with control calluses, as well as lower membrane degradation. These data show that calluses are more tolerant than plants to Cd exposure, and that antioxidant mechanisms under Cd exposure may differ between the two cell systems and vary with time.     

Electron paramagnetic resonance studies of manganese toxicity,tolerance, and amelioration with silicon in snapbean

Plant genotypes within species differ widely in tolerance to excess manganese (Mn) that may occur in acid soils, or in neutral or alkaline soils having poor aeration caused by imperfect drainage or compaction. However, Mn tolerance mechanisms in plants are largely unknown. Silicon (Si) is reported to detoxify Mn within plants, presumably by preventing localized accumulations of Mn associated with lesions on leaves. Because Mn is paramagnetic, electron paramagnetic resonance (EPR) spectroscopy, shows promise as a tool for characterizing toxic and non‐toxic forms of Mn in tolerant and sensitive plants. The objective of our study was to use EPR to: i) determine the chemical/ physical state of Mn in Mn‐tolerant and ‐sensitive snapbean cultivars; and ii) characterize the protective effects of Si against Mn toxicity. Manganese‐sensitive Wonder Crop 2 (WC) and Mn‐tolerant Green Lord (GL) cultivars of snapbean were grown at pH 5.0, in a greenhouse, in a modified Steinberg solution containing: Mn=0.05mg.L^‐1 (optimal); Mn=1.0mgL^‐1 (toxic); Mn=1.0 mg L^‐1 plus Si=4 mg L^‐1; and Mn=0.05 mg L^‐1 plus 4 mg Si L^‐1. All trifoliate leaf samples exhibited a 6‐line EPR signal that is characteristic of hexaaquo Mn²⁺. In both cultivars, a higher EPR Mn²⁺ signal‐intensity generally correlated with lower total leaf mass, higher total Mn concentrations and more pronounced symptoms of toxicity. Tolerance to excess Mn coincided with lower Mn²⁺ signal intensity. Silicon treatments ameliorated Mn toxicity symptoms in both genotypes, decreased total leaf Mn concentrations, and decreased EPR Mn²⁺ signal intensity. Results suggest that Mn toxicity is associated with reduced electron transport and accumulation of oxidation products in leaves. Amelioration of Mn toxicity by Si is regarded as connected with a reduction in this Mn‐induced process. Results indicated that EPR spectroscopy can be useful in investigating the biochemical basis for differential Mn tolerance in plants. The EPR observations might also help plant breeders in developing Mn‐tolerant cultivars.     

Cadmium Uptake and Distribution by Different Maize Genotypes in Maturing Stage

Abstract

A pot experiment was conducted to investigate the effects of different cadmium (Cd) concentrations of phaeozem on growth and uptake of Cd and mineral nutrient copper (Cu) and zinc (Zn) by three maize genotypes in the mature stage. The results showed that the dry‐matter accumulation of shoots was inhibited by added Cd for Jidan209 and Jitian6, but this did not influence Chunyou30. The root biomasses decreased significantly for Jitian6 and stimulated Jidan209 and Chunyou30. Yields of three genotypes of maize were decreased by increasing soil Cd concentrations. Among them, Chunyou30 had a high tolerance and Jitian6 was most sensitive to Cd. The accumulation order of Cd in different parts of plants was root > leaf > stem > grain. The percentage of absorbed Cd by roots was 70–85% of total absorbed amount. Cadmium uptake by maize in the mature stage had a significant genetic variation: Jitian6 > Jidan209 > Chunyou30 for root, stem and leaf, and Jidan209 > Jitian6 > Chunyou30 for grain, respectively. Increase of soil Cd had no significant effect on Zn concentration of leaves, but there was a significant genetic variation: Chunyou30 > Jidan209 > Jitian6 (P=0.023). Cu concentration of leaves was increased significantly with increase of soil Cd (P<0.01), but no genetic variation was observed.     

Cadmium‐induced changes in glutathione and phenolics of Thlaspi and Noccaea species differing in Cd accumulation

Glutathione (GSH) and phenolics play an important role in plant defense against metal‐ion toxicity. The antioxidant activity and metal‐binding capacity of these compounds can account for the protective effects. In contrast to animal‐cell models, however, the possible interplay among these substances in stress defense of plants is poorly investigated. This study compares the influence of cadmium (Cd) on the profiles of both soluble phenolics and GSH in shoots of different Thlaspi and Noccaea species: two ecotypes of the nonhyperaccumulator T. arvense differing in Cd resistance (ecotype Aigues Vives, Cd‐sensitive, and ecotype Jena, Cd‐resistant) and two Cd‐tolerant Cd‐Zn hyperaccumulators N. praecox and N. caerulescens (formerly Thlaspi praecox and T. caerulescens). To reveal the possible influence of Cd‐induced sulfur (S) shortage on the stress response, plants receiving normal S concentrations (500 μM MgSO₄) and plants treated with surplus S (500 μM MgSO₄ + 500 μM K₂SO₄) were analyzed. Our working hypothesis was that species differences in tolerance to high tissue Cd concentrations should be reflected by differences in endogenous levels of GSH and phenolic compounds. The results reveal clear species‐dependent differences in both the constitutive patterns and the Cd‐ and S‐induced changes in shoot concentrations of GSH and phenolics. However, no simple relationship between these shoot concentrations and Cd accumulation and tolerance can be established.     

丛枝菌根真菌对万寿菊生长、吸镉及生理特性的影响

A pot experiment was carried out to study the effects of three arbuscular mycorrhizal fungi (AMF), including Glomus intraradices, Glomus constrictum and Glomus mosseae, on the growth, root colonization and Cd accumulation of marigold (Tagetes erecta L.) at Cd addition levels of 0, 5 and 50 mg kg-1 in soil. The physiological characteristics, such as chlorophyll content, soluble sugar content, soluble protein content and antioxidant enzyme activity, of Tagetes erecta L. were also investigated. The symbiotic relationship between the marigold plant and arbuscular mycorrhizal fungi was well established under Cd stress. The symbiotic relationship was reffected by the better physiobiochemical parameters of the marigold plants inoculated with the three AMF isolates where the colonization rates in the roots were between 34.3% and 88.8%. Compared with the non-inoculated marigold plants, the shoot and root biomass of the inoculated marigold plants increased by 15.2%- 47.5% and 47.8%-130.1%, respectively, and the Cd concentration and accumulation decreased. The chlorophyll and soluble sugar contents in the mycorrhizal marigold plants increased with Cd addition, indicating that AMF inoculation helped the marigold plants to grow by resisting Cd stress. The antioxidant enzymes reacted differently with the three AMF under Cd stress. For plants inoculated with G. constrictum and G. mosseae, the activities of superoxide dismutase (SOD) and catalase (CAT) increased with increasing Cd addition, but peroxidase (POD) activity decreased with increasing Cd addition. For plants inoculated with G. intraradices, three of the antioxidant enzyme activities were significantly decreased at high levels of Cd addition. Overall, the activities of the three antioxidant enzymes in the plants inoculated with AMF were higher than those of the plants without AMF inoculation under Cd stress. Our results support the view that antioxidant enzymes have a great influence on the biomass of plants, and AMF can improve the capability of reactive oxygen species (ROS) scavenging and reduce Cd concentration in plants to alleviate Tagetes erecta L. from Cd stress.     

Mycorrhizal fungi and earthworms reduce antioxidant enzyme activities in maize and sunflower plants grown in Cd-polluted soils

Changes in plant antioxidant enzymes (AOEs) in response to cadmium (Cd) pollution are an important mechanism for plant growth and tolerance to Cd-induced stress. The main objective of this greenhouse study was to determine the combined influence of earthworm and arbuscular mycorrhiza (AM) fungal inoculation and their interactions with Cd on AOEs and proline accumulation in leaves of two major crops under Cd stress. Maize (Zea mays L.) and sunflower (Helianthus annuus L.) plants were exposed to Cd stress (10 and 20 mg kg⁻¹ soil), inoculated with either earthworm (Lumbricus rubellus L.) or AM fungi (Glomus intraradices and Glomus mosseae species) in a pot experiment for three months. Exposure to Cd decreased shoot dry weights, increased shoot Cd and P concentrations, leaf proline accumulation and the activity of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and polyphenol oxidase (PPO) in both mycorrhizal and non-mycorrhizal plants and both in the presence and absence of earthworms. Inoculation of both model plants with earthworms and AM fungi decreased shoot Cd concentrations and the activity of all AOEs, except PPO. Although earthworm activity enhanced the proline content of sunflower in Cd-polluted soils, the proline level of both plants remained unaffected by AM fungi. AM fungi and earthworms may decrease the activity of AOEs through a decline in shoot Cd toxicity and concentration, confirming that plant inoculation with these soil organisms improves maize and sunflower tolerance and protection against Cd toxicity. Generally, the effect of AM fungal inoculation on plant responses to Cd addition was greater than that of earthworm activity. Nonetheless, the interactive effect of AM fungus and earthworm is of minor importance for most of the plant AOEs in Cd-polluted soils.