Materials

The rice variety used in this study was Mianhui 6139, a hybrid rice restorer line developed by the Mianyang Academy of Agricultural Sciences, China. In May 2023, rice seeds were sown in trays filled with perlite and placed in a greenhouse under the conditions described by Li et al. [21]. After germination, the trays were irrigated with Hoagland solution every three days. When the rice seedlings reached an approximate height of 10 cm (three-leaf stage), they were transplanted into square pots.

GABA was obtained from Beijing Solarbio Science and Technology Co. Ltd. (Beijing, China).

Experimental Design

The experiment was conducted in a rain shelter between June and July 2023. Uniform rice seedlings at the three-leaf stage were transplanted into square pots (height, 6.5 cm; length, 11 cm; and width, 8 cm) filled with perlite, with four seedlings evenly distributed in the corners of each pot. Six treatments were applied in this experiment: (1) control (CK), without Cd treatment;

(2) 1 mg/L Cd treatment (Cd); (3) 1 mg/L Cd treatment + 0.1 mmol/L GABA (GABA0.1); (4) 1 mg/L Cd treatment + 0.25 mmol/L GABA (GABA0.25);

(5) 1 mg/L Cd treatment + 0.5 mmol/L GABA (GABA0.5); and (6) 1 mg/L Cd treatment + 1 mmol/L GABA (GABA1) [22]. Hoagland nutrient solution containing 1 mg/L Cd (in the form of CdCl₂·2.5H₂O) and GABA was applied to the pots for the Cd, GABA0.1, GABA0.5, GABA1, and GABA2 treatments, whereas Hoagland nutrient solution without Cd and GABA was applied for the CK treatment. Each treatment was replicated thrice, with one pot serving as the replicate. All Hoagland nutrient solutions were replaced every three days. After one month of growth, all plants were harvested to determine various parameters.

Determination of Parameters

In July 2023, the third mature leaf from the top of the main stem was selected to measure the photosynthetic characteristics using a Li-6400 photosynthetic system (LI-COR, Lincoln, NE, USA). The parameters assessed included the net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO₂ concentration (Ci), and transpiration rate (Tr). The conditions for determining these photosynthetic parameters were established according to a previous report [21]. The same leaves were collected to evaluate the levels of photosynthetic pigments (chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid) and the activities of antioxidant enzymes, such as peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD). The levels of photosynthetic pigments were measured using the acetone-ethanol mixed extraction method [23]. Antioxidant enzyme activity was determined using previously described methods [23, 24]. The whole plants were harvested, washed, and separated into roots, stems and sheaths, and leaves. The tissue samples were dried at 80ºC until they reached a constant weight to record their dry weight (biomass). Dried tissue samples were finely ground and digested in a mixture of nitric and perchloric acids. The resulting solution was analyzed for Cd content using an iCAP 6300 ICP-MS spectrometer (Thermo Fisher Scientific, Waltham, MA, USA).

Statistical Analysis

SPSS software version 20.0.0 (IBM, Inc., Armonk, NY, USA) was used for all statistical analyses. Data were analyzed using a one-way analysis of variance and Duncan’s multiple range test (p<0.05). Pearson’s correlation analysis was used to assess correlations among all parameters under Cd stress.

Biomass of Rice Seedlings Under Cd Stress

Under Cd stress, rice growth was inhibited because of the toxic effects of Cd [25]. In this study, the Cd treatment decreased rice seedlings’ biomass compared with CK (Table 1). Compared with CK, the Cd treatment decreased the root, stem and sheath, and leaf biomass by 14.35%, 14.48%, and 13.54%, respectively, indicating that treatment with 1 mg/L Cd inhibited rice growth, which aligns with the findings of a previous study [25].

Under Cd stress, GABA could enhance biomass and promote maize growth [26]. GABA, at appropriate concentrations, can mitigate the inhibitory effects of Cd stress on plant height and stimulate growth [17, 18, 22]. In this study, the concentrations of 0.1, 0.25, and 0.5 mmol/L GABA increased the root, stem and sheath, and leaf biomass of rice seedlings under Cd stress, whereas GABA at a concentration of 1 mmol/L only increased the stem and sheath biomass (Table 1). Compared with the Cd treatment, the concentrations of 0.1, 0.25, and 0.5 mmol/L GABA increased the root biomass by 65.64%, 48.88%, and 38.83%, respectively; stem and sheath biomass by 21.43%, 17.99%, and 15.08%, respectively; and leaf biomass by 32.21%, 29.70%, and 19.55%, respectively. The concentration of 0.1 mmol/L GABA was the best dose, which increased the root and leaf biomass by 41.87% and 14.30%, respectively, compared with CK. These results suggest that GABA can promote rice growth under Cd stress, consistent with previous studies [17, 18, 22, 26]. This effect may be attributed to GABA acting as a signaling molecule that enhances plant growth by influencing the expression and activity of key enzymes (e.g., photosynthetic enzymes) and ion channel-related proteins, thereby facilitating the uptake of essential mineral elements and improving plant resistance [27].

Photosynthetic Pigment Content in Rice Seedlings Under Cd Stress

Photosynthetic pigments are the core components of the photosynthetic system in higher plants and significantly influence the photosynthesis rate, reflecting plants’ physiological evolution [28]. Cd stress inhibits the biosynthesis of photosynthetic pigments in plants [15, 16]. In this study, compared with CK, the Cd treatment decreased photosynthetic pigment content in rice seedlings (Table 2). Compared with CK, the Cd treatment decreased the contents of chlorophyll a, chlorophyll b, and total chlorophyll by 20.28%, 22.60%, and 20.77%, respectively. Treatment with 1 mg/L Cd inhibited the biosynthesis of photosynthetic pigments in rice, consistent with previous studies [15, 16].

Under Cd stress, GABA increased the content of photosynthetic pigments in oilseed rape, indicating that GABA promotes the biosynthesis of these pigments [16]. GABA also enhances chlorophyll a, chlorophyll b, and carotenoid levels in Camptotheca acuminata seedlings under Cd stress [15]. In this study, the concentrations of 0.1, 0.25, and 0.5 mmol/L GABA increased the contents of chlorophyll a, chlorophyll b, and total chlorophyll in rice seedlings under Cd stress, whereas the concentration of 1 mmol/L GABA did not affect these parameters (Table 2). Compared with the Cd treatment, the concentrations of 0.1, 0.25, and 0.5 mmol/L GABA increased the chlorophyll a by 34.04%, 30.16%, and 20.81%, respectively; chlorophyll b content by 36.00%, 22.80%, and 20.80%, respectively; and total chlorophyll by 34.41%, 28.78%, and 20.81%, respectively. For carotenoid content, only the concentrations of 0.1 and 0.25 mmol/L GABA resulted in significant increases in the carotenoid content, whereas the concentrations of 0.5 and 1 mmol/L GABA did not have any effect. The concentration of 0.1 mmol/L GABA was the best dose, which increased the contents of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid by 6.86%, 5.26%, 6.49%, and 11.43%, respectively, compared with CK. Therefore, GABA may promote the biosynthesis of photosynthetic pigments in rice under Cd stress to some extent, which aligns with previous findings [15, 16]. This effect may be attributed to the ability of GABA to mitigate stress-induced cell membrane damage and facilitate membrane recovery [29, 30]. Further investigation is required to elucidate the underlying mechanisms of action.

Photosynthetic Characteristics of Rice Seedlings Under Cd Stress

Under Cd stress, photosynthesis in crops is impaired, leaf stomatal resistance increases, and stomatal density decreases [31]. In this study, the Cd treatment reduced the rice seedlings’ Pn, Gs, Ci, and Tr by 13.16%, 27.72%, 11.00%, and 21.02%, respectively, compared with CK (Table 3). This result indicates that treatment with 1 mg/L Cd inhibited photosynthesis in rice.

The application of GABA can enhance the Pn, Tr, and Gs in plant leaves, thereby improving photosynthesis [32]. In this study, 0.1 and 0.25 mmol/L GABA concentrations increased the Pn of rice seedlings under Cd stress, whereas the concentrations of 0.5 and 1 mmol/L GABA had no significant effects (Table 3). The Gs and Ci values treated with GABA at concentrations of 0.1, 0.25, and 0.5 mmol/L were higher than those under Cd treatment, whereas treatment with 1 mmol/L GABA showed no significant effect. The Tr values for all four GABA concentrations were higher than those for the Cd treatment. Overall, treatment with 0.1 mmol/L GABA yielded the most significant improvements in the Pn, Gs, Ci, and Tr, which increased by 14.77%, 32.44%, 11.61%, and 21.37%, respectively, compared with the Cd treatment. These results align with those of previous studies on cucumber [33] and oilseed rape [16], suggesting that GABA can mitigate the inhibitory effects of Cd stress on photosynthesis and likely influences stomatal opening in rice by regulating photosynthetic processes [34].

Antioxidant Enzyme Activity of Rice Seedlings Under Cd Stress

The concentration of ROS in crops increases under Cd stress, leading to the elevated activity of antioxidant enzymes that scavenge excessive ROS [35, 36]. In this study, the Cd treatment enhanced the activities of POD, CAT, and SOD in rice seedlings by 13.21%, 4.03%, and 5.71%, respectively, compared with CK (Table 4). This finding is consistent with those of previous studies [37]. These results indicated that treatment with 1 mg/L Cd caused oxidative damage to rice, prompting an increase in antioxidant enzyme activity to adapt to Cd stress.

GABA plays a crucial role in regulating the antioxidant defense system of crops, thereby alleviating adverse stress conditions [26, 38]. Under salinity stress, foliar application of 4-6 mmol/L GABA significantly increased the activities of SOD, POD, and CAT in oat seedlings [12]. In the presence of Cd stress, GABA reduced root CAT and POD activities in Festuca elata, decreased shoot POD activity, and increased shoot CAT activity [39]. In this study, concentrations of 0.5 and 1 mmol/L GABA enhanced the POD activity of rice seedlings by 17.05% and 54.95%, respectively, compared with the Cd treatment (Table 4). The 0.1 mmol/L GABA concentration did not affect the POD activity, whereas the 0.25 mmol/L GABA concentration resulted in a decrease compared with the Cd treatment. Additionally, 0.1 and 0.25 mmol/L GABA concentrations reduced the CAT activity, whereas the 0.5 and 1 mmol/L GABA concentrations increased the CAT activity compared with the Cd treatment. For the SOD activity, only the 1 mmol/L GABA concentration led to an increase, whereas the other GABA concentrations resulted in a decrease compared with the Cd treatment. These findings are not entirely consistent with those observed in oats and F. elata [12, 29], suggesting that GABA may enhance antioxidant enzyme activity in rice to a certain extent. This effect may be attributed to the ability of GABA to upregulate the expression of antioxidant enzyme genes in plants [40], which helps scavenge ROS and mitigate the toxic effects of Cd on plants [29].

Cd Content in Rice Seedlings Under Cd Stress

Under chromium (Cr) stress, GABA decreased the Cr content in the roots and shoots of mustard, alleviated the toxicity of Cr to mustard, and promoted mustard growth [41]. Similarly, under Cd stress, GABA decreased Cd content in oilseed rape and limited the accumulation of Cd in its organelles, thereby alleviating the toxicity associated with Cd stress [16]. In hyperaccumulator plants, GABA increased Cd content in S. nigrum var. humile [33]. In this study, the root Cd content in rice seedlings ranged from 91.89 to 154.63 mg/kg, whereas the Cd content in the leaves was between 19.25 and 47.55 mg/kg, and the leaf content ranged from 3.37 to 15.89 mg/kg under Cd stress (Table 5). Compared with the Cd treatment, the concentrations of 0.1 and 0.25 mmol/L GABA did not significantly affect or even decrease the root Cd content, whereas the concentrations of 0.5 and 1 mmol/L GABA increased it. The concentrations of 0.1 and 0.25 mmol/L GABA decreased the Cd content in the stem and sheath by 29.51% and 17.03%, respectively, whereas the higher concentrations of 0.5 and 1 mmol/L GABA led to an increase in these values compared with the Cd treatment. Additionally, concentrations of 0.1, 0.25, 0.5, and 1 mmol/L GABA reduced leaf Cd content by 78.79%, 71.74%, 56.77%, and 28.82%, respectively, compared with the Cd treatment. These results suggest that low GABA concentrations may inhibit Cd accumulation in rice, whereas high concentrations can promote Cd accumulation. This effect may be attributed to the ability of GABA to alter the subcellular distribution of elements and mitigate ROS induced by Cd stress [42-44].

Correlation Analysis

Correlation analysis was used to assess correlations among all parameters under Cd stress. The root, stem and sheath, and leaf biomass were positively correlated with one another (Table 6) and with chlorophyll a content, chlorophyll b content, total chlorophyll content, carotenoid content, Pn, Gs, Ci, and Tr. Conversely, root, stem, sheath, and leaf biomass were negatively correlated with SOD activity and leaf Cd content. Additionally, root biomass was negatively correlated with POD activity, CAT activity, root Cd content, and Cd content in both the stems and sheaths. Similarly, stem and sheath biomasses were negatively correlated with CAT activity. The contents of root Cd and stem and sheath Cd were positively correlated with each other, negatively correlated with chlorophyll a content, total chlorophyll content, carotenoid content, Pn, Gs, and Ci, and positively correlated with POD and CAT activities. Furthermore, leaf Cd content was negatively correlated with chlorophyll a content, chlorophyll b content, total chlorophyll content, carotenoid content, Pn, Gs, Ci, and Tr but positively correlated with CAT and SOD activities.