Comprehensive Physio-Biochemical Evaluation Reveals Promising Genotypes and Mechanisms for Cadmium Tolerance in Tibetan Hull-Less Barley

Abstract

Cadmium (Cd) toxicity in agricultural soil is increasing globally and significantly impacts crop production and food safety. Tibetan hull-less barley (Hordeum vulgare L. var. nudum), an important staple food and economic crop, exhibits high genetic diversity and is uniquely adapted to the harsh conditions of the Qinghai-Tibet Plateau. This study utilized hydroponic experiments to evaluate the genotypic differences in Cd tolerance among 71 Tibetan hull-less barley genotypes. Physiological assessments revealed significant reductions in various growth parameters under Cd stress compared to normal conditions: soil-plant analysis development (SPAD) value, shoot height, root length, shoot and root fresh weight, shoot and root dry weight, of 11.74%, 39.69%, 48.09%, 52.88%, 58.39%, 40.59%, and 40.52%, respectively. Principal component analysis (PCA) revealed key traits contributing to Cd stress responses, explaining 76.81% and 46.56% of the variance in the preliminary and secondary selection. The genotypes exhibited varying degrees of Cd tolerance, with X178, X192, X215, X140, and X162 showing high tolerance, while X38 was the most sensitive based on the integrated score and PCA results. Validation experiments confirmed X178 as the most tolerant genotype and X38 as the most sensitive, with observed variations in morphological, physiological, and biochemical parameters, as well as mineral nutrient responses to Cd stress. Cd-tolerant genotypes exhibited higher chlorophyll content, net photosynthesis rates, and effective photochemical capacity of photosystem II, along with an increased Cd translocation rate and reduced oxidative stress. This was accompanied by elevated activities of antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), indicating a robust stress response mechanism. These findings could facilitate the development of high-tolerance cultivars, with X178 as a promising candidate for further research and cultivation in Cd-contaminated soils.

Keywords: antioxidant enzyme; cadmium tolerance; hull-less barley; integrated score; oxidative stress; photosynthesis.

Figure 1

Differences in growth traits and integrated scores among 71 barley varieties under Cd stress. (A–G) Percentage reduction in various growth parameters after 15 days of exposure to 20 µM Cd stress compared to control conditions. (H) Integrated score based on these growth parameters; The growth parameters of barley seedlings were assessed as a percentage of the control to evaluate the impact of Cd stress. FW = fresh weight, DW = dry weight. ● Tolerant, ● sensitive, ○ not considered for further evaluation. Data are presented as means of three biological replicates (n = 3). The inset “|” indicates the least significant difference (LSD) at the 0.05 probability level between varieties.

Figure 2

Differences in growth traits and integrated scores among the seven barley genotypes. (A–G) Percentage reduction in seven growth traits after 10 days of exposure to 20 µM Cd stress, expressed as a percentage of the control values. (H) Integrated scores for each genotype. FW = fresh weight; DW = dry weight. ■ Tolerant genotypes, ■ sensitive genotype, ■ check genotype (a Cd-tolerant reported previously [22]). Data are presented as means ± SD (n = 3). One-way ANOVA was used, and multiple comparisons were made using Duncan’s test. Different letters indicate significant differences at p < 0.05.

Figure 3

Bi-plot based on principle component analysis of reduction percentage of barley seedling morphological characters under 20 µM Cd stress conditions. (A) Preliminary selection (15 days after treatment), (B) secondary selection (10 days after treatment). (SPAD = SPAD value, SH = shoot height, RL = root length, SFW = shoot fresh weight, RFW = root fresh weight, RDW = root dry weight and SDW = shoot dry weight, IS = integrated score).

Figure 4

Phenotypical observation of X178, Weisuobuzhi, and X38 under control and 20 µM Cd stress (10 days after treatment, 15 days after germination). Differences in growth traits of tolerant genotype (X178), check genotype (Weisuobuzhi), and sensitive genotype (X38) varieties after 15 days under control and 20 µM Cd stress. (A–F) Six growth traits. FW = fresh weight, DW = dry weight. Data are presented as means ± SD (n = 3). One-way ANOVA was used, and multiple comparisons were made using Duncan’s test. Different letters indicate significant differences at p < 0.05.

Figure 5

Effects of photosynthesis parameters of the tolerant genotype (X178), check genotype (Weisuobuzhi), and sensitive genotype (X38) under control and 20 µM Cd stress. (A) SPAD value; (B) net photosynthetic rate, Pn; (C) stomatal conductance, Gs; (D) intercellular carbon dioxide concentration, Ci; (E) transpiration rate, Tr; (F) effective photochemical efficiency of photosystem II, PhiPS2. Data are presented as means ± SD (n = 3). One-way ANOVA was used, and multiple comparisons were made using Duncan’s test. Different letters indicate significant differences at p < 0.05.

Figure 6

Cd content in shoot (A) and root (B) of barley seedlings after 15 days of 20 µM Cd treatment. DW, dry weight. Translocation factor = Cd concentration in shoot/Cd concentration in the root (C). Data are presented as means ± SD (n = 3). One-way ANOVA was used, and multiple comparisons were made using Duncan’s test. Different letters indicate significant differences at p < 0.05.

Figure 7

Effects of Cd stress on the concentrations of Zn, Cu, Mn, and Fe (mg kg⁻¹ dry weight) in shoot (A–D) and root (E–H) of barley seedlings after 15 days of 20 µM Cd treatment. Data are presented as means ± SD (n = 3). One-way ANOVA was used, and multiple comparisons were made using Duncan’s test. Different letters indicate significant differences at p < 0.05.

Figure 8

Effects of Cd on contents of malondialdehyde (MDA (A)), hydrogen peroxide (H₂O₂ (B)), and antioxidant enzyme activities of SOD (C), POD (D), and CAT (E) of leaves in barley seedlings after 10 days of Cd treatment. Data are presented as means ± SD (n = 3). One-way ANOVA was used, and multiple comparisons were made using Duncan’s test. Different letters indicate significant differences at p < 0.05.

Figure 9

Effects of Cd toxicity on morpho-physiological, elemental (shoot and root), oxidative, and antioxidant (leaves) parameters of barley. Net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular carbon dioxide concentration (Ci), transpiration rate (Tr), effective photochemical efficiency of photosystem II (PhiPS2), malondialdehyde (MDA), hydrogen peroxide (H₂O₂), superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). Each parameter changes in measured parameters under Cd treatment compared to the control.