Exogenous γ‐polyglutamic acid alleviates cadmium stress in rice through redox regulation and modulation of cadmium accumulation

Abstract

BACKGROUND

Cadmium (Cd) contamination severely threatens crop production and irreversibly damages plant growth, yield, and quality. The purpose of the present study was to investigate the effect of γ‐polyglutamic acid (γ‐PGA) on Cd tolerance in rice during the whole growth period.

METHODS

The experiment consisted of four treatments: a control group with neither γ‐PGA nor Cd; a group treated with γ‐PGA alone; a group treated with Cd alone; and a group treated with both γ‐PGA and Cd.

RESULTS

Results showed that γ‐PGA significantly alleviated Cd stress by promoting rice growth and immobilizing bioavailable Cd in soil. Physiological analysis showed that chlorophyll and shoot proline peaked at the tillering stage, while root proline peaked at the heading stage. Malondialdehyde (MDA) and H₂O₂ increased with growth, with higher MDA in roots. Antioxidant enzymes showed distinct patterns. Superoxide dismutase and root catalase peaked at the tillering stage, shoot peroxidase at the heading stage. Under Cd stress, chlorophyll synthesis was markedly inhibited, oxidative damage was induced, and the antioxidant defense system was activated. γ‐PGA alone had no significant effect, whereas Cd + γ‐PGA co‐treatment effectively mitigated Cd toxicity by increasing chlorophyll, antioxidant enzyme activities, and proline accumulation, as well as reducing oxidative damage, with the most pronounced effects at the tillering and maturity stages. Principal component analysis further indicated that the alleviative role of γ‐PGA was growth stage‐specific.

CONCLUSION

Research findings, therefore, suggest that γ‐PGA can ameliorate Cd toxicity in rice, resulting in improved plant growth under metal stress. © 2026 Society of Chemical Industry.

INTRODUCTION

In recent years, cadmium (Cd) contamination in paddy soils has become an increasing environmental concern, drawing increasing attention from the scientific community. ¹ The global average Cd concentration in rice ranges from 100.41 to 173.00 μg kg⁻¹, exceeding the safety standard of 0.1 mg kg⁻¹ (equivalent to 100 μg kg⁻¹) established by the Food and Agriculture Organization of the United Nations and the World Health Organization. ² The global issue of Cd in rice primarily stems from Cd contamination in agricultural soils. ³ According to statistical data, the total global area of agricultural land contaminated with trace elements reaches 2.35 × 10¹² m², among which approximately 2.78 × 10⁹ m² of agricultural soil in China is contaminated with Cd. ⁴ In China, the acceleration of industrialization and the extensive use of agricultural chemicals have led to a concerning trend of soil Cd pollution, which remains far from being effectively controlled. ⁵ The annual increase in soil Cd levels, at a rate of 0.004 mg kg⁻¹ a⁻¹, significantly surpasses the average growth rate observed in European soils. ⁶ Cadmium in soil exists in multiple chemical forms that determine its uptake rate and accumulation in rice, with exchangeable Cd (primarily as free Cd²⁺ ions) being the most bioavailable and mobile fraction responsible for Cd stress in paddy soils and rice plants due to its high absorption potential. ⁷ Rice actively absorbs free Cd²⁺ from soil, with significant accumulation occurring in roots. ⁸ The metal is subsequently transported to root vascular cylinders via both apoplastic and symplastic pathways. ⁹ Through the activity of tonoplast‐localized transporters, Cd²⁺ is loaded into xylem vessels and then translocated to aerial parts via transpiration‐driven flow and root pressure. Ultimately, Cd accumulates in panicles during grain filling, entering the human food chain through rice consumption and posing direct health risks.^{10, 11, 12}

γ‐Polyglutamic acid (γ‐PGA) is a microbial‐derived amino acid polymer composed of d‐ and l‐glutamate units linked through amide bonds between α‐amino and γ‐carboxyl groups, featuring abundant highly reactive free carboxyl side chains along its molecular backbone. ¹³ γ‐PGA effectively chelates free metal ions through its abundant carboxyl functional groups, converting them from free ionic states to stable chelated forms. ¹⁴ This biopolymer exhibits exceptional flocculation capacity in efficient adsorption of heavy metal contaminants in both soils and aqueous solutions. ¹⁵ As an environmentally compatible material, γ‐PGA demonstrates high solubility and biodegradable characteristics, preventing secondary pollution in remediation applications. ¹⁶ Its nontoxic nature renders γ‐PGA highly promising for environmental remediation purposes. ¹⁷ Studies have demonstrated that γ‐PGA exhibits excellent adsorption capacity for heavy metal ions in aqueous systems. However, the influence of γ‐PGA on plant heavy metal uptake in soil environments presents greater complexity. Due to the unique properties of soil systems and variations in plant–soil interactions, γ‐PGA exerts dual effects: as a polymeric organic acid, root application promotes the dissolution and release of lead, thereby enhancing lead accumulation in crops; however, excessive concentrations inhibit plant growth and reduce fresh biomass. Conversely, foliar application significantly decreases Cd uptake in pepper plants (Capsicum annuum).^{18, 19} These contrasting outcomes primarily depend on application methods and plant species specificity.

Despite these advancements, the specific mechanisms by which γ‐PGA mitigates Cd toxicity in rice remain poorly understood. Therefore, the study reported here aimed to explore the role of γ‐PGA in alleviating Cd toxicity in rice, with a focus on its effects on growth, physiological parameters, and antioxidant defense systems. By elucidating the underlying mechanisms, this research will provide valuable insights into the development of sustainable strategies for mitigating Cd contamination in rice production systems.

MATERIALS AND METHODS

Growth conditions and applied treatments

The commonly used rice cultivar ‘Fengliangyou4’ from China was employed as the model plant in this study. Rice seeds were soaked and then cultivated in a nutrient solution at 28 °C in a light incubator until the two‐leaf‐one‐heart stage, after which they were transplanted into the treated soil. A pot cultivation method was adopted in this experiment, with four replicates arranged, and 20 seedlings were planted in each replicate pot. The test clayey soil was collected from ordinary farmland in Wuhan, Hubei Province. The concentration of γ‐PGA (sodium polyglutamate, purity > 99.9%; CAS: 84960‐48‐5; molecular weight: 387.34g mol⁻¹; Junan Biotechnology, China) in the test soil was set to 0.6 g kg⁻¹ (kg referring to the weight of the soil). The selection of this concentration was based on preliminary Cd absorption kinetic experiments. Nitrogen, phosphorus, and potassium fertilizers were supplied at normal application levels. The tested soil had a Cd concentration of 3 mg kg⁻¹, achieved by adding CdCl₂·2.5H₂O, with the calculation based on the elemental Cd content. The selection of this Cd treatment concentration was based on its status as a typical concentration in rice Cd stress research, which induces moderate to severe Cd stress without plant death, making it suitable for whole‐growth‐period physiological index determination. The same concentration of NPK fertilizer (0.3 g N kg⁻¹ soil, 0.15 g P₂O₅ kg⁻¹ soil, 0.15 g K₂O kg⁻¹) was applied to each pot of soil. The air‐dried soil, sieved through a 2 mm mesh, was placed in plastic containers of 8 kg each, for a total of 16 pots. The experiments included the following four treatments of rice seedlings: (i) no γ‐PGA and Cd (control, CK); (ii) 0.6 g kg⁻¹ γ‐PGA‐treated soil; (iii) 3 mg kg⁻¹ Cd‐treated soil; (iv) 0.6 g kg⁻¹ γ‐PGA‐ and 3 mg kg⁻¹ Cd‐treated soil.

Morphological measurements and yield characters

The rice plants were harvested at the seedling, tillering, heading, and maturity stages, and the morphological data were collected on the same day for three randomly selected plants from each treatment. Shoot and root lengths were measured in uprooted plant samples. The root fresh weight (RFW), shoot fresh weight (SFW), and total fresh weight (TFW = RFW + SFW) were measured using a digital weighing balance. The fresh weight samples were then oven‐dried at 70 °C for 72 h to afford the root dry weight (RDW), shoot dry weight (SDW), and total dry weight (TDW = RDW + SDW). At the mature stage, the plants from five pots of every treatment were harvested at 85 days after transplanting, including rice shoots and roots. Roots were cleaned with tap water, and then all plants were rinsed with deionized water at least three times. After being dried with blotting paper, all plants were divided into four parts: roots, stems (including panicle branch), leaves, and grains. The grains were air‐dried and weighed. Yield components were characterized as ears per pot, spikelets per ear, percentage of filled spikelets, and 1000‐grain weight from 10 plants sampled randomly from each pot. Roots, stems, and leaves were first dried in an oven at 110 °C for 30 min, and then dried at 70 °C to constant weight and weighed on a ten‐thousandth scale.

Cd contents and translocation factors

Approximately 0.2 g of ground plant and soil samples was weighed into a digestion vessel, and the digestion was performed using nitric acid, followed by overnight standing. The digestion vessel was then placed in a microwave digestion system and digested for 45 min. After cooling, the samples were diluted to 50 mL, and the total Cd content was determined using an atomic absorption spectrophotometer (contrAA 700, Analytik Jena, Germany) at the appropriate wavelength. The bioconcentration factor (BCF) in plant parts was calculated as Cd contents (plant part)/Cd contents (medium), while the translocation factor (TF) was computed as Cd contents in shoot/Cd contents in root. ²⁰

Pigment contents

The samples for all biochemical analyses were collected at different growth stages. To determine leaf chlorophyll a and b (Chl a and Chl b) and total chlorophyll (total Chl) contents, approximately 50 mg of fresh leaf sample was extracted with ethanol and stored in the dark for 12 h. The absorbances were recorded with a UV–visible spectrophotometer (A590, AOELAB, Shanghai, China) at 663 and 645 nm wavelengths, and the pigment contents (mg g⁻¹ FW) were calculated following the formula described by Lichtenthaler. ²¹

MDA, H₂O₂ , and proline contents

The malondialdehyde (MDA) contents in the samples were measured by TBARS (thiobarbituric acid reactive substance) assay. ²² Fresh samples (0.2 g) were ground and extracted with ethanol for 12 h. The supernatant was mixed with TBA reagent and heated in a boiling water bath. After cooling, the absorbance was measured at 532 nm. The proline contents were estimated by the modified acid ninhydrin colorimetric method described by Carillo et al. ²³ with minor modification. ²⁴ The optical density of the prepared samples was taken at 520 nm, and the proline content (μg g⁻¹ FW) was quantified from a standard curve of l‐proline created with known concentrations. The H₂O₂ content was determined using a hydrogen peroxide assay kit (Solarbio, Beijing, China), following the manufacturer's protocol.

Assay of enzymatic activities

To determine the activities of enzymatic antioxidants such as superoxide dismutase (SOD; EC1.15.1.1), catalase (CAT; EC1.11.1.6), and peroxidase (POD; EC1.11.1.7), approximately 0.2 g of sample was pulverized in a pre‐cooled mortar and pestle with phosphate buffer, and the supernatant was collected after centrifugation. The activity of the SOD enzyme was assessed using the nitroblue tetrazolium colorimetric method as described by Beauchamp and Fridovich. ²⁵ The CAT and POD enzymatic activities were determined following the protocols developed by Aebi ²⁶ and Chance and Maehly. ²⁷

Statistical data analysis

We repeated all experiments at least three times in independent experiments. Results were analyzed via one‐way analyses of variance (ANOVAs) and Duncan's multiple range test was carried out to test significant differences at P < 0.05. All data were expressed as mean ± standard deviation.

RESULTS

Effect of γ‐PGA on rice growth period under Cd stress

Cadmium stress and its amelioration through exogenous γ‐PGA application significantly altered the growth period of rice (Table 1). The seedling stage in the Cd treatment was prolonged by 1 day compared to CK, while the γ‐PGA + Cd treatment maintained the same duration as CK. The tillering stage in Cd treatment was notably delayed by 7 and 9 days relative to CK and γ‐PGA treatment, respectively; however, γ‐PGA + Cd treatment shortened this period to 30 days, similar to γ‐PGA treatment. The heading stage and maturation stage in Cd treatment showed slight prolongation, but Cd + γ‐PGA treatment kept them the same as for CK. For the total growth period, Cd treatment extended it to 147 days, while Cd + γ‐PGA treatment reduced it to 136 days, consistent with CK and γ‐PGA treatment.

Table 1.

Effects of γ‐PGA on rice growth period

Stage	CK	γ‐PGA	Cd	Cd + γ‐PGA
Germination stage (d)	7 a	7 a	7 a	7 a
Seedling stage (d)	24 a	24 a	25 a	24 a
Tillering stage (d)	30 b	28 b	37 a	30 b
Heading stage (d)	8 a	8 a	9 a	8 a
Maturation stage (d)	67 a	67 a	69 a	67 a
Total (d)	136 b	134 b	147 a	136 b

Letters ‘a’, ‘b’, ‘c’, ‘d’ indicate the difference between different treatments, P < 0.05.

Effect of γ‐PGA on rice growth under Cd stress

Cadmium stress and its amelioration with exogenous γ‐PGA application significantly altered the morphological traits of rice (Table 2). Compared with CK, γ‐PGA application led to a remarkable increase in all growth parameters at every developmental stage. At the seedling stage, RFW, SFW, TFW, RDW, SDW, and TDW in the γ‐PGA group increased by 131.4%, 239.0%, 122.0%, 134.3%, 108.8%, and 130.1%, respectively. During the tillering stage, the above parameters in the γ‐PGA group were 239.0%, 206.6%, 224.3%, 220.0%, 207.9%, and 217.2% higher than those in the CK group. At the heading stage, the increases in RFW, SFW, TFW, RDW, SDW, and TDW in the γ‐PGA group compared to CK were 82.7%, 99.1%, 103.1%, 107.1%, 170.8%, and 114.2%, respectively. At the maturation stage, RFW, SFW, TFW, RDW, SDW, and TDW in the γ‐PGA group were 21.2%, 142.0%, 44.1%, 18.4%, 153.5%, and 33.3% higher than those in the CK group. Under Cd stress, γ‐PGA application effectively alleviated the growth inhibition caused by Cd across all developmental stages. At the seedling stage, compared with the Cd‐alone group, RFW, SFW, TFW, RDW, SDW, and TDW in the Cd + γ‐PGA group increased by 109.9%, 99.8%, 85.8%, 62.4%, 76.9%, and 53.4%, respectively. At the tillering stage, SFW, TFW, RDW, SDW, and TDW in the Cd + γ‐PGA group were 96.8%, 98.9%, 83.7%, 59.6%, and 78.2% higher than those in the Cd‐alone group. At the heading stage, the increases in TFW, RDW, SDW, and TDW in the Cd + γ‐PGA group compared to the Cd‐alone group were 68.5%, 73.5%, 55.8%, and 71.6%, respectively. At the maturation stage, RDW, SDW, and TDW in the Cd + γ‐PGA group were 81.4%, 35.3%, and 74.6% higher than those in the Cd‐alone group. The results demonstrated that γ‐PGA application substantially enhanced plant growth under both CK and Cd stress, with this promoting effect being consistent across all developmental stages.

Table 2.

Effects of γ‐PGA on RFW, SFW, TFW, RDW, SDW, and TDW

Stage		CK	γ‐PGA	Cd	Cd + γ‐PGA
Seedling stage	RFW	0.2906 ± 0.0007 c	0.6724 ± 0.0125 a	0.2239 ± 0.0080 d	0.4699 ± 0.0070 b
	SFW	2.2880 ± 0.0075 c	7.7571 ± 0.0077 b	2.1587 ± 0.003 a	4.3134 ± 0.009 a
	TFW	0.3230 ± 0.0008 c	0.7169 ± 0.0135 a	0.2907 ± 0.005 d	0.5402 ± 0.0041 b
	RDW	0.0461 ± 0.004 c	0.1080 ± 0.0007 a	0.0375 ± 0.002 d	0.0609 ± 0.0020 b
	SDW	0.0091 ± 0.0001 b	0.0190 ± 0.0004 a	0.0065 ± 0.004 b	0.0115 ± 0.0002 b
	TDW	0.0552 ± 0.0005 c	0.1270 ± 0.0010 a	0.0472 ± 0.0002 d	0.0724 ± 0.0018 b
Tillering stage	RFW	2.2880 ± 0.0075 c	7.7571 ± 0.0077 a	2.0587 ± 0.0031 d	4.3134 ± 0.0093 b
	SFW	1.0486 ± 0.0078 c	3.2155 ± 0.1842 a	0.9864 ± 0.0917 c	1.9414 ± 0.0107 b
	TFW	3.3366 ± 0.0151 c	10.8199 ± 0.1920 a	3.1451 ± 0.0888 c	6.2548 ± 0.0168 b
	RDW	0.4234 ± 0.0018 c	1.3548 ± 0.0059 a	0.4117 ± 0.0004 d	0.7564 ± 0.0063 b
	SDW	0.1289 ± 0.0003 c	0.3969 ± 0.0017 a	0.1219 ± 0.0001 d	0.1946 ± 0.0006 b
	TDW	0.5523 ± 0.0020 c	1.7517 ± 0.0073 a	0.5336 ± 0.0003 d	0.9510 ± 0.0068 b
Heading stage	RFW	43.9400 ± 0.2006 c	80.3000 ± 2.4554 a	38.3833 ± 0.0235 d	67.6500 ± 0.8774 b
	SFW	12.7333 ± 0.2239 c	25.3462 ± 0.0169 a	11.6500 ± 0.1485 d	16.6633 ± 0.0094 b
	TFW	56.6733 ± 0.1793 c	115.1233 ± 2.4721 a	50.0333 ± 0.1621 d	84.3133 ± 0.8818 b
	RDW	12.2380 ± 0.4046 c	25.3462 ± 0.0256 a	11.3076 ± 0.0054 d	19.6145 ± 0.0326 b
	SDW	1.5279 ± 0.0057 c	4.1369 ± 0.2019 a	1.3512 ± 0.0079 c	2.1047 ± 0.0043 b
	TDW	13.7659 ± 0.4103 c	29.4832 ± 0.1903 a	12.6588 ± 0.0134 d	21.7195 ± 0.0297 b
Maturation stage	RFW	52.2333 ± 0.6852 b	63.3300 ± 0.2502 a	31.7300 ± 0.0081 c	57.3266 ± 3.2786 b
	SFW	13.6630 ± 0.4076 b	33.0648 ± 0.0448 a	11.6100 ± 0.0081 c	14.7365 ± 1.2669 b
	TFW	66.8964 ± 0.2833 b	96.3948 ± 0.2735 a	43.3400 ± 0.0081 c	72.0632 ± 4.4869 b
	RDW	17.5753 ± 04129 b	20.8117 ± 0.5039 a	10.4943 ± 0.0630 c	19.0416 ± 1.0455b
	SDW	2.1737 ± 0.1120 bc	5.5107 ± 0.0074 a	1.8150 ± 0.1546 c	2.4560 ± 0.2111 b
	TDW	19.7490 ± 0.4869 b	26.3225 ± 0.5081 a	12.3093 ± 0.1833 c	21.4977 ± 1.2454 b

All data are presented as mean ± standard deviation (n ≥ 3). Significant differences were analyzed by one‐way ANOVA followed by Duncan's multiple range test (P < 0.05). Values with different lowercase letters indicate significant differences among treatments at the same growth stage.

Effect of γ‐PGA on yield under Cd stress

Exogenous γ‐PGA application altered rice growth and grain traits at the maturation stage under Cd stress (Fig. 1). Compared to CK, Cd stress reduced tiller number by 20.3%, grain width by 3.7%, grain length by 5.7%, 1000‐kernel weight by 8.7%, seed‐setting rate by 14.5%, and single‐plant yield by 22.2%, while plant height showed no significant difference among treatments. Application of γ‐PGA alone increased tiller number by 47.8%, 1000‐kernel weight by 5.1%, and seed‐setting rate by 10.7% compared to CK, with plant height, grain width, and grain length remaining stable. Notably, the Cd + γ‐PGA combined treatment effectively mitigated Cd‐induced growth and yield inhibition. Relative to the Cd‐stressed plants, Cd + γ‐PGA treatment increased tiller number by 43.7%, grain width by 0.8%, grain length by 2.9%, 1000‐kernel weight by 6.3%, seed‐setting rate by 18.8%, and single‐plant yield by 27.7%, with plant height still showing no significant difference compared to CK and Cd‐stress treatments.

Figure 1.

Effects of γ‐PGA on (A, D) grain width, (B, E) grain length, (C) plant height, (F) single‐plant yield, (G) tiller number, (H) 1000‐kernel weight, and (I) seed‐setting rate. Means followed by the same letter are not significantly different within the variety (P < 0.05).

Effect of γ‐PGA on chlorophyll content in rice under Cd stress

The exogenous application of γ‐PGA increased the chlorophyll content under stress (Fig. 2). At the seedling stage, Cd stress significantly reduced Chl a, Chl b, and total Chl content by 16.8%, 18.8%, and 17.6% respectively, while γ‐PGA alone increased these parameters by 2.0%, 3.3%, and 2.6% respectively, and the Cd + γ‐PGA treatment mitigated the Cd‐induced reduction. At the tillering stage, γ‐PGA application exerted a prominent effect: Chl a, Chl b, and total Chl content in the γ‐PGA group were 8.9%, 19.3%, and 14.1% higher, while Cd stress caused a 6.1%, 4.3%, and 5.2% decrease; the Cd + γ‐PGA treatment effectively alleviated this decline. During the heading stage, Cd stress led to a severe reduction in chlorophyll content, whereas γ‐PGA alone increased these parameters by 7.1%, 23.5%, and 10.4% respectively, and the Cd + γ‐PGA treatment restored chlorophyll levels. At the maturation stage, Cd stress decreased Chl a, Chl b, and total Chl content by 16.7%, 48.9%, and 24.6% respectively, while the Cd + γ‐PGA treatment mitigated this reduction, with chlorophyll content increased compared to the Cd‐alone group. Collectively, these results demonstrate that γ‐PGA application can significantly alleviate the Cd‐induced reduction in chlorophyll content across all growth stages.

Figure 2.

Effects of γ‐PGA on (A) Chl a, (B) Chl b, and (C) total Chl. Values are given as the means of three biological replicates (n = 3), with bars indicating ±SE. Bars with different letters indicate significant differences according to Tukey comparison test (P < 0.05).

Effect of γ‐PGA on MDA, H₂O₂ , and proline contents in rice under Cd stress

To comprehensively evaluate the role of γ‐PGA in modulating osmotic adjustment and oxidative stress responses in rice under Cd stress, we assessed proline content (Fig. 3(A), shoot; Fig. 3(B), root), MDA content (Fig. 3(C), shoot; Fig. 3(D), root), and hydrogen peroxide (H₂O₂) content (Fig. 3(E), shoot; Fig. 3(F), root) across seedling, tillering, heading, and maturation stages. At the seedling stage, Cd stress triggered a marked accumulation of osmotic and oxidative stress markers, with shoot proline, root proline, shoot MDA, root MDA, shoot H₂O₂, and root H₂O₂ significantly increased compared with CK, while exogenous γ‐PGA application – either alone or in combination with Cd (Cd + γ‐PGA) – effectively counteracted these increases, with the Cd + γ‐PGA treatment reducing shoot proline by 13.8%, root proline by 56.9%, shoot MDA by 13.9%, root MDA by 22.81%, shoot H₂O₂ by 43.8%, and root H₂O₂ by 30.4% relative to the Cd‐alone group. During the tillering stage, Cd stress exerted a more pronounced impact, increasing shoot proline content, root proline content, shoot MDA content, root MDA content, shoot H₂O₂ content, and root H₂O₂ content all significantly compared with CK; γ‐PGA alone significantly elevated proline, MDA, and H₂O₂ levels compared to CK, likely reflecting its role in priming stress tolerance, and the Cd + γ‐PGA treatment demonstrated robust alleviation, reducing shoot proline by 11.2%, root proline by 39.6%, shoot MDA by 15.9%, root MDA by 21.4%, shoot H₂O₂ by 19.2%, and root H₂O₂ by 29.2% relative to the Cd‐alone group. At the heading stage, Cd stress again induced significant increases in these parameters, and γ‐PGA application attenuated these responses, with the Cd + γ‐PGA group showing reductions of 13.4–46.1% in proline, 21.8–55.2% in MDA, and 22.9–34.0% in H₂O₂ across shoots and roots relative to the Cd‐alone group. In the maturation stage, compared with CK, Cd stress increased shoot proline content, root proline content, as well as the MDA and H₂O₂ contents in both shoots and roots. In contrast, the Cd + γ‐PGA treatment reduced shoot proline content by 11.4%, root proline content by 41.3%, shoot MDA content by 17.9%, root MDA content by 46.1%, shoot H₂O₂ content by 9.9%, and root H₂O₂ content by 39.6% relative to the Cd‐alone group. Additionally, the proline, MDA, and H₂O₂ contents in the γ‐PGA‐only group were comparable to those in CK, suggesting that γ‐PGA has the potential to enhance long‐term stress resilience in mature plants.

Figure 3.

Effects of γ‐PGA on (A, B) proline, (C, D) MDA, and (E, F) hydrogen peroxide contents. Values are given as the means of three biological replicates (n = 3), with bars indicating ±SE. Bars with different letters indicate significant differences (P ≤ 0.05) according to Tukey comparison test.

Cd content, BCF, and TF

To comprehensively assess the role of γ‐PGA in regulating Cd accumulation and translocation in rice under Cd stress, we examined Cd content in shoots (Fig. 4(A)) and roots (Fig. 4(B)), Cd content in rice‐growing soil (Fig. 4(C)), BCF in roots (Fig. 4(D)) and shoots (Fig. 4(E)), and TF (Fig. 4(F)) across seedling, tillering, heading, and maturation stages. At the seedling stage, Cd stress triggered substantial Cd accumulation in rice, with shoot Cd content reaching 10.5 mg kg⁻¹ DW and root Cd content surging to 19.2 mg kg⁻¹ DW. Exogenous γ‐PGA application – alone or in combination with Cd – robustly inhibited Cd uptake. The Cd + γ‐PGA group showed 66.5% and 60.2% decreases in shoot and root Cd content, respectively, 19.9% higher soil Cd content (indicating reduced absorption), and 66.8%, 72.0%, and 15.7% decreases in root BCF, shoot BCF, and TF, respectively, compared to the Cd‐alone group. During the tillering stage, Cd‐induced shoot and root Cd accumulation reached 5.6 and 14.8 mg kg⁻¹ DW, respectively. γ‐PGA treatment alleviated these increases, with the Cd + γ‐PGA group exhibiting 66.1% and 37.5% decreases in shoot and root Cd content, 58.9% higher soil Cd content, and 60.8%, 78.7%, and 45.7% decreases in root BCF, shoot BCF, and TF, respectively. At the heading stage, Cd stress increased shoot and root Cd content to 1.9 and 13.8 mg kg⁻¹ DW, respectively. The Cd + γ‐PGA treatment reduced these values by 60.8% and 32.8%, elevated soil Cd content by 89.9%, and decreased root BCF, shoot BCF, and TF by 29.0%, 79.5%, and 71.1%, respectively. In the maturation stage, shoot and root Cd contents in the Cd group were 0.7 and 12.1 mg kg⁻¹ DW, respectively. The Cd + γ‐PGA treatment decreased these by 36.7% and 47.3%, increased soil Cd content by 209.0%, and reduced root BCF, shoot BCF, and TF by 36.3%, 80.2%, and 68.9%, respectively. Collectively, γ‐PGA significantly inhibits Cd accumulation in rice shoots and roots, reduces Cd uptake from the soil, and decreases Cd bioconcentration and translocation across all growth stages, highlighting its potential as a biostimulant to mitigate Cd toxicity by modulating Cd uptake and translocation processes.

Figure 4.

Effects of γ‐PGA on (A) shoot Cd content, (B) root Cd content, (C) soil Cd content, (D, E) BCF, and (F) TF. Values are given as the means of three biological replicates (n = 3), with bars indicating ±SE. Bars with different letters indicate significant differences (P ≤ 0.05) according to Tukey comparison test.

Effect of γ‐PGA on enzymatic antioxidant activities in rice under Cd stress

To investigate the effects of γ‐PGA on antioxidant enzyme activities in rice under Cd stress, we analyzed SOD (Fig. 5(A),(B)), POD (Fig. 5(C),(D)), and CAT (Fig. 5(E),(F)) activities in shoots and roots across seedling, tillering, heading, and maturation stages. At the seedling stage, Cd stress significantly increased these enzyme activities, and γ‐PGA application – alone or with Cd – further enhanced them, with the Cd + γ‐PGA group showing the highest levels: shoot SOD activity was 345.6 U min⁻¹ g⁻¹ FW and 11.5% higher than Cd; root SOD activity was 209.1 U min⁻¹ g⁻¹ FW and 6.2% higher than Cd; shoot POD activity was 46.7 nmol L⁻¹ min⁻¹ g⁻¹ FW and 16.5% higher than Cd; root POD activity was 36.3 nmol L⁻¹ min⁻¹ g⁻¹ FW and 58.8% higher than CK; shoot CAT activity was 78.9 nmol L⁻¹ min⁻¹ g⁻¹ FW and 34.5% higher than Cd; root CAT activity was 140.5 nmol L⁻¹ min⁻¹ g⁻¹ FW and 20.5% higher than Cd. During the tillering stage, Cd stress continued to induce high activities, and γ‐PGA treatment maintained elevated levels: shoot SOD activity was 456.9 U min⁻¹ g⁻¹ FW and 19.7% higher than Cd; root SOD activity was 231.2 U min⁻¹ g⁻¹ FW and 5.4% higher than CK; shoot POD activity was 51.2 nmol L⁻¹ min⁻¹ g⁻¹ FW and 16.2% higher than Cd; root POD activity was 30.0 nmol L⁻¹ min⁻¹ g⁻¹ FW and 91.3% higher than Cd; shoot CAT activity was 134.8 nmol L⁻¹ min⁻¹ g⁻¹ FW and 49.8% higher than Cd; root CAT activity was 207.4 nmol L⁻¹ min⁻¹ g⁻¹ FW and 11.6% higher than Cd. At the heading stage, Cd stress still increased enzyme activities, and γ‐PGA alleviated stress while keeping activities high: shoot SOD activity was 337.1 U min⁻¹ g⁻¹ FW and 12.4% higher than Cd; root SOD activity was 212.2 U min⁻¹ g⁻¹ FW and 3.9% higher than Cd; shoot POD activity was 79.5 nmol L⁻¹ min⁻¹ g⁻¹ FW and 20.4% higher than Cd; root POD activity was 5.1 nmol L⁻¹ min⁻¹ g⁻¹ FW and 45.9% higher than Cd; shoot CAT activity was 82.2 nmol L⁻¹ min⁻¹ g⁻¹ FW and 41.0% higher than Cd; root CAT activity was 166.6 nmol L⁻¹ min⁻¹ g⁻¹ FW and 15.0% higher than Cd. In the maturation stage, Cd stress‐induced activities remained notable, and γ‐PGA maintained relatively high levels: shoot SOD activity was 327.5 U min⁻¹ g⁻¹ FW and 25.9% higher than Cd; root SOD activity was 172.7 U min⁻¹ g⁻¹ FW and 2.8% higher than Cd; shoot POD activity was 34.7 nmol L⁻¹ min⁻¹ g⁻¹ FW and 54.8% higher than Cd; root POD activity was 3.3 nmol L⁻¹ min⁻¹ g⁻¹ FW and 29.6% higher than Cd; shoot CAT activity was 54.6 nmol L⁻¹ min⁻¹ g⁻¹ FW and 18.3% higher than Cd; root CAT activity was 96.7 nmol L⁻¹ min⁻¹ g⁻¹ FW and 15.7% higher than Cd. Collectively, γ‐PGA enhances SOD, POD, and CAT activities in rice under Cd stress across all growth stages, indicating its potential to improve antioxidant defense.

Figure 5.

Effects of γ‐PGA on activities of (A, B) SOD, (C, D) POD, and (E, F) CAT. Values are given as the means of three biological replicates (n = 3), with bars indicating ±SE. Bars with different letters indicate significant differences (P ≤ 0.05) according to Tukey comparison test.

Principal component analysis (PCA)

To investigate the multivariate relationships among measured traits under different growth stages and treatments, we performed PCA‐biplot analysis for rice plants at seedling, tillering, heading, and maturation stages (Fig. 6), with the biplots constructed based on the first two principal components (PCs) as they captured the highest percentage of total variation across all stages. At the seedling stage, PC1 accounted for 74.9% of total variability, primarily associated with root/shoot H₂O₂, MDA contents, fresh/dry weights, and chlorophyll traits, while PC2 was associated with proline content and antioxidant enzyme activities, with γ‐PGA and Cd + γ‐PGA groups distinctly separated from the Cd‐alone group. During the tillering stage, PC1 (68.5% of variation) strongly correlated with root/shoot Cd contents, proline, and biomass traits, and PC2 associated with chlorophyll parameters, showing clear separation between Cd‐alone and γ‐PGA/Cd + γ‐PGA treatments. At the heading stage, PC1 (65.1% of variation) was linked to root/shoot Cd, proline, and biomass‐related traits, PC2 to chlorophyll and antioxidant enzyme activities, and γ‐PGA/Cd + γ‐PGA groups diverged from Cd alone. In the maturation stage, PC1 (66.0% of variation) was associated with root/shoot Cd, proline, biomass, and chlorophyll traits, PC2 with H₂O₂, MDA, and antioxidant enzyme activities, and Cd‐alone treatment clustered distinctly from γ‐PGA and Cd + γ‐PGA treatments. Collectively, γ‐PGA application significantly altered the multivariate relationships among physiological and growth traits under Cd stress, with PC1 consistently dominating total variability via Cd accumulation, biomass, and chlorophyll traits, and PC2 reflecting variance in antioxidant and proline‐related responses across all growth stages of rice.

Figure 6.

PCA‐biplot displaying the variability among the measured 32 traits from six growth conditions in Oryza sativa L.

DISCUSSION

Cadmium, a nonessential yet profoundly toxic heavy metal, exhibits significant bioaccumulative potential within trophic chains and demonstrates remarkable persistence in human tissues, with an estimated biological half‐life ranging from 20 to 30 years.^{28, 29} Designated as a Group 1 human carcinogen by the International Agency for Research on Cancer, this metallic element poses significant health threats through its cumulative accumulation in biological systems. Rice is a globally significant staple cereal crop that exhibits marked heavy metal accumulation characteristics, showing the highest Cd bioaccumulation capacity among cereal crops. ³⁰ In recent years, the application of exogenous γ‐PGA to alleviate heavy metal toxicity in plants has garnered significant scientific attention. Therefore, this study assessed the effectiveness of γ‐PGA application at different growth stages in mitigating Cd toxicity in rice plants, through a comprehensive investigation of growth parameters, physiological characteristics, and redox regulation systems.

γ‐PGA mitigates Cd‐induced growth inhibition and restores normal growth period dynamics

Plant growth is substantially impaired by Cd stress through disruption of cell division, inhibition of nutrient element/ion absorption, suppression of photosynthesis, and exacerbation of oxidative stress.^{31, 32} The results of this study demonstrated that Cd exerted significant inhibitory effects on plant growth, as evidenced by reduced fresh and dry biomass accumulation in both shoots and roots, decreased tiller number, and rediced yield (Table 2, Fig. 1). Notably, exogenous application of γ‐PGA significantly ameliorated these Cd‐induced adverse effects on rice growth, which aligns with previous findings on γ‐PGA's protective roles in cucumber and pepper.^{18, 33} Unlike previous studies focusing on single growth stages or specific organs, our data highlight that γ‐PGA's growth‐promoting effect is consistent across the entire rice life cycle – from seedling to maturation stages. For instance, at the tillering stage (a critical period for biomass accumulation), the Cd + γ‐PGA treatment increased shoot fresh weight by 68.5% and total dry weight by 78.2% compared to the Cd‐alone group (Table 2), underscoring its sustained protective capacity.

As a nonessential element for plants, Cd, in the form of Cd²⁺, can enter plant tissues and displace essential metal ions such as Zn²⁺, Mg²⁺, and Fe²⁺ from their binding sites in metalloproteins, thereby disrupting ionic homeostasis. ³⁴ γ‐PGA enhances the bioavailability of cationic nutrients such as Ca, Mg, Cu, and Zn in the soil through the adsorption and activation capacity of the ionized carboxyl groups on its side chains. ³⁵ This improved nutrient availability drives the concurrent promotion of lateral root growth and tillering in rice, thereby leading to a significant increase in overall biomass. ³⁶ Our findings further revealed that γ‐PGA application alleviates Cd‐induced prolongation of the growth period (Table 1), particularly shortening the tillering stage of Cd‐stressed rice from 37 days to 30 days. This restoration of developmental rhythm likely stems from γ‐PGA's dual role in maintaining nutrient homeostasis and sequestering Cd – preventing Cd from interfering with cell cycle progression and hormonal regulation of phenological stages.^{37, 38, 39} Rice plants exhibit a higher Cd uptake rate during the tillering stage, and our data confirm that γ‐PGA effectively chelates Cd ions at this critical stage, reducing Cd absorption efficiency. ⁴⁰ Additionally, the excellent water retention capacity of γ‐PGA helps improve the rhizosphere microenvironment, indirectly alleviating Cd‐induced oxidative stress and ensuring normal developmental processes during tillering.^{36, 41}

γ‐PGA restores photosynthetic pigment content and maintains photosynthetic function under Cd stress conditions

The suppression of plant growth by Cd is directly reflected in concomitant alterations in photosynthesis and photosynthetic pigments.^{3, 42} In this study, Cd stress significantly reduced Chl a, Chl b, and total Chl content at all growth stages – with the most severe reduction observed at the tillering stage (Fig. 2). This Cd‐induced chlorophyll loss may be attributed to two key mechanisms. First, Cd inhibits the activity of enzymes involved in chlorophyll biosynthesis. Second, Cd‐induced reactive oxygen species (ROS) accumulation damages chloroplast ultrastructure and accelerates chlorophyll degradation. ⁴³ Exogenous γ‐PGA application effectively reversed this trend: at the heading stage (when photosynthetic demand is highest), the Cd + γ‐PGA treatment restored total chlorophyll content to CK levels, significantly higher than the 20.9% observed in the Cd‐alone group (Fig. 2(C)). This protective effect of γ‐PGA on photosynthetic pigments may be associated with the finding by Mu et al. ³⁷ that γ‐PGA exhibits a binding efficiency of 51% for water‐soluble calcium (Ca) and magnesium (Mg). Since Mg is a key element for chlorophyll synthesis, γ‐PGA likely promotes chlorophyll production by enhancing the bioavailability of Mg. Specifically, γ‐PGA may stabilize chlorophyll synthase and prevent its degradation by Cd ions, while also reducing ROS‐mediated damage to chloroplasts – ultimately maintaining chlorophyll homeostasis and ensuring efficient light energy capture for photosynthesis.

γ‐PGA enhances antioxidant defense systems to alleviate Cd‐induced oxidative damage

The primary adverse consequence of heavy metal stress is the excessive generation of ROS, including O²⁻, H₂O₂, and •OH, which subsequently results in oxidative damage to plants. ⁴⁴ Rather than participating directly in free radical production, Cd counteracts enzymatic and nonenzymatic antioxidants, thereby disturbing the balance between ROS and the antioxidant defense system. ⁴⁵ In this study, Cd stress significantly increased H₂O₂ and MDA content in rice shoots and roots – at the seedling stage, root MDA content in the Cd‐alone group was twice that in the CK group (Fig. 3(D)), indicating severe lipid peroxidation and membrane damage. Exogenous γ‐PGA application effectively mitigated this oxidative stress: the Cd + γ‐PGA treatment reduced shoot H₂O₂ content by 43.8% and root MDA content by 22.8% at the seedling stage, and these protective effects persisted through maturation (Fig. 3(C)–(F)).

This alleviation of oxidative damage by γ‐PGA is mediated through both enzymatic and nonenzymatic antioxidant pathways. Proline, a key nonenzymatic antioxidant, accumulates in response to Cd stress as a protective mechanism – regulating osmotic potential, stabilizing membrane structures, and scavenging ROS.^{46, 47} Our data showed that Cd stress increased root proline content at the seedling stage, and γ‐PGA further enhanced this accumulation (Fig. 3(B)). However, in the Cd + γ‐PGA group, proline content was lower than in the Cd‐alone group at later growth stages, which likely reflects reduced ROS production (and thus reduced demand for proline) due to γ‐PGA's Cd‐chelating effect. For enzymatic antioxidants, γ‐PGA significantly enhanced the activity of SOD, CAT, and POD under Cd stress. Among these, POD – an enzyme that scavenges H₂O₂ by catalyzing the oxidation of guaiacol – exhibited the most pronounced response: at the tillering stage, shoot POD activity in the Cd + γ‐PGA group was 16.2% higher than in the Cd‐alone group (Fig. 5(C)). This increase in POD activity, combined with elevated SOD (which dismutates O₂ ⁻ to H₂O₂) and CAT (which decomposes H₂O₂ to H₂O and O₂), forms a synergistic antioxidant network efficiently quenching excess ROS. Notably, γ‐PGA alone had no significant effect on antioxidant enzyme activity relative to CK, indicating that its regulatory role in the antioxidant system is stress‐dependent – specifically activated under Cd‐induced oxidative stress.

γ‐PGA inhibits Cd uptake and translocation to reduce Cd accumulation in rice

Our findings revealed that γ‐PGA significantly reduced Cd accumulation in rice shoots and roots across all growth stages. At the seedling stage, the Cd + γ‐PGA treatment decreased shoot and root Cd content by 66.5% and 60.2%, respectively, compared to the Cd‐alone group (Fig. 4(A),(B)). This reduction in Cd accumulation is primarily driven by two mechanisms. First, γ‐PGA's abundant carboxyl groups chelate free Cd²⁺ in the soil, converting it into a stable, non‐bioavailable form. ¹⁵ This is supported by our data showing that soil Cd content in the Cd + γ‐PGA group was 20.0% higher than in the Cd‐alone group at the seedling stage (Fig. 4(C)), indicating reduced Cd absorption by rice roots. Second, γ‐PGA competes with Cd²⁺ for cation transporters in root cell membranes, reducing Cd²⁺ entry into root cells.^{48, 49} This is reflected in the lower BCF in the Cd + γ‐PGA group: root BCF was reduced by 66.8% at the seedling stage, and shoot BCF by 72.2% (Fig. 4(D),(E)), confirming reduced Cd uptake efficiency.

In addition to inhibiting Cd uptake, γ‐PGA also reduces Cd translocation from roots to shoots. The TF in the Cd + γ‐PGA group was 15.7% lower than in the Cd‐alone group at the seedling stage (Fig. 4(F)), and this trend persisted through maturation. This restricted translocation may be due to enhanced Cd sequestration in root vacuoles and binding to root cell walls – two key detoxification mechanisms in plants.^{50, 51, 52} By limiting Cd translocation to aerial parts (especially shoots and grains), γ‐PGA not only reduces Cd‐induced oxidative damage (as reflected by lower MDA and H₂O₂ levels) but also minimizes Cd entry into the human food chain. This is consistent with Wang et al., ⁵³ who reported that γ‐PGA reduces Cd transfer from stems to leaves and ears in rice, enabling normal plant function under Cd stress.

Developmental stage‐specificity of γ‐PGA's protective effects

A key insight from this study is the developmental stage‐specificity of γ‐PGA's role in alleviating Cd stress. PCA (Fig. 6) revealed that PC1 (accounting for 65.1–74.9% of total variation across stages) was consistently linked to Cd accumulation, biomass, and chlorophyll traits, while PC2 reflected variance in antioxidant and proline‐related responses – highlighting dynamic shifts in γ‐PGA's regulatory focus. At the seedling stage, γ‐PGA primarily enhances antioxidant defense and reduces Cd uptake, as seedlings are most vulnerable to oxidative damage and Cd‐induced growth arrest. During the tillering stage, γ‐PGA shifts to promoting root growth and tillering while maintaining Cd chelation – supporting the establishment of a robust root system to cope with subsequent Cd stress. At the heading and maturation stages, γ‐PGA's focus transitions to limiting Cd translocation to shoots and grains and maintaining photosynthetic function – ensuring sufficient photoassimilate accumulation for grain filling. This stage‐specific regulation allows γ‐PGA to efficiently allocate resources to the most critical stress–response processes at each developmental phase, maximizing its protective effect.

Integration of γ‐PGA's mechanisms of Cd stress alleviation

In summary, collectively, our results support a multifaceted mechanism by which γ‐PGA alleviates Cd stress in rice (Fig. 7). (i) Cd chelation and uptake inhibition: γ‐PGA chelates free Cd²⁺ in soil and competes for root transporters, reducing Cd accumulation. (ii) Translocation restriction: γ‐PGA enhances Cd sequestration in roots, reducing Cd movement to shoot parts. (iii) Growth promotion: γ‐PGA improves nutrient availability and water retention, promoting root growth and tillering. (iv) Photosynthetic protection: γ‐PGA stabilizes chlorophyll content, maintaining light energy capture. (v) Oxidative stress mitigation: γ‐PGA enhances antioxidant enzyme activity and proline accumulation, scavenging excess ROS and reducing membrane damage. Importantly, γ‐PGA disrupts the ‘Cd accumulation–ROS production–plant damage’ positive feedback loop, with this effect persisting across the entire growth period.

Figure 7.

Model representing γ‐PGA‐mediated changes for regulation of Cd stress in Oryza sativa L.

Compared to previous studies, our work advances understanding of γ‐PGA's role in Cd stress alleviation by: (i) investigating its effects across the full rice growth cycle, rather than single stages; (ii) revealing its stage‐specific regulatory mechanisms; and (iii) integrating physiological (growth, photosynthesis) and molecular (antioxidant enzymes, Cd transporters) responses. These findings highlight γ‐PGA's potential as an environmentally friendly biostimulant for mitigating Cd contamination in rice production systems, offering a sustainable alternative to chemical soil amendments.

CONCLUSIONS

As a biological chelating agent, γ‐PGA alleviates Cd stress in rice through dual mechanisms: the carboxyl groups on its molecular side chains chelate free Cd²⁺ ions, reducing Cd uptake and accumulation in plants, while its regulation of physiological metabolism decreases MDA content and increases both proline content and total antioxidant capacity to maintain cellular homeostasis. This protective effect demonstrates developmental stage‐specific dynamics, shifting from predominant oxidative defense during the seedling stage to focused control of shoot Cd translocation in later stages. The study systematically reveals the effectiveness of γ‐PGA in mitigating heavy metal stress through synergistic actions, offering new solutions for farmland heavy metal remediation and safe crop production.

CONFLICT OF INTEREST

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

AUTHOR CONTRIBUTIONS

Rong Huang: Data curation, Investigation, Writing – original draft, First author. Qiang Qiang: Data curation, Investigation, First author. Zhichao Wang: Data curation, Investigation. Changhuan Du: Investigation. Junxing Tan: Investigation. Xiangyu Huang: Investigation. Xuezhu Du: Conceptualization, Supervision, Writing – review & editing. Feng Sheng: Conceptualization, Supervision, Writing – review & editing.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.