Effects of cadmium stress on seed germination and physiological-biochemical characteristics in okra: a comparative study of red and green varieties

Determination of germination rate and germination potential

Seeds of red and green okra (purchased from Hebei Nanjixing Seed Company) were sterilized in 3% H₂O₂ for 15 min, rinsed thrice with distilled water, and blotted dry. Cd solutions (0, 0.5, 1.0, 1.5, 2.0 mM) were prepared by diluting 0.1 M CdCl₂•2.5H₂O stock. For 2.0 mM solution, 40 mL of stock was diluted to 2 L with distilled water. Each 9-cm-diameter petri dish (lined with two layers of filter paper) was saturated with a Cd solution. Thirty seeds were placed per dish (n = 3 replicates per treatment) and incubated at 24 °C in darkness. The number of germinated seeds was recorded each day for 7 days (germination was defined as radicle emergence ≥ 2 mm) and calculated the seed germination potential and germination rate. The formula for the germination potential (GP) is given by the following equation, ${\displaystyle \mathrm{GP}\left(\text{\%}\right)=\left(\frac{\mathrm{G}3}{\mathrm{Gn}}\right)\times 100}$

where GP is the 3-day germination count (G3) divided by the tested count (Gn). And the formula for the germination rate (GR) is given by the following equation, ${\displaystyle \mathrm{GR}\left(\text{\%}\right)=\left(\frac{\mathrm{G}7}{\mathrm{Gn}}\right)\times 100}$

where GR is the 7-day germination count (G7) divided by Gn.

Plant growth and treatment

The seeds germinated in a plastic container (length 28 cm, width 20 cm, height 10 cm) with vermiculite. Water was added once daily to keep the vermiculite moist. The 7-day-old seedlings were transferred to 1-litre flasks filled with Hoagland’s nutrient solution, with 10 plants in each. After 3 days, the Hoagland nutrient solution in pots was replaced by a new nutrient solution containing 0 or 50 µM Cd (added as CdCl₂⋅2.5H₂O). The choice of 50 µM Cd was based on previous research on Cd stress in Vicia sativa (Rui et al., 2016), where this concentration was found to be effective in inducing measurable physiological and biochemical responses without causing extreme toxicity that could mask specific stress-related effects. Plant growth and treatment experiments were all conducted in a greenhouse at 26 ± 2 °C, 70–80% relative humidity and 14 h photoperiod.

Biomass determination

After 6 days of Cd exposure, plant phenotypes were observed and photographed, and lengths of longest roots and shoots were recorded. The roots and shoots of the seedlings were collected separately. The tissues were thoroughly rinsed with deionized water three times and blotted dry with absorbent paper. For moisture content determination, fresh weight (FW) was immediately measured using an analytical balance after surface moisture removal. Samples were then oven-dried at 80 °C for 24 h until constant weight was achieved. Dry weight (DW) was recorded after cooling samples in a desiccator containing silica gel for 30 min. Moisture content (%) was calculated as: [(FW–DW)/FW] ×100. Each plastic cup contained ten seedlings as a replicate. Three plastic cups, or 30 seedlings, were randomly selected for one treatment.

Determination of Cd content

Roots and shoots were harvested separately and baked at 80 °C for 24 h. The dried samples were ground into powder and passed through a 60-mesh sieve (with a pore size of approximately 0.25 mm). Five milliliters of nitric acid and 0.1 mL of internal standard mix were added to 200 mg of powder. After 1 h, the mixture was diluted to 25 mL and then digested according to the standard procedure of the microwave digestion apparatus. The digestion process was carried out by gradually raising the temperature to 120 °C and maintaining it for 5 min, followed by further increasing the temperature to 160 °C and holding it for 10 min, and finally elevating the temperature to 180 °C and maintaining it for 20 min. After completion of the digestion process, the samples cooled to room temperature. The samples were determined by inductively coupled plasma mass spectrometry (ICP-MS). For quality assurance, a blank solution and certified Cd were analyzed after every ten samples to verify the accuracy and precision of the measurements. The internal standard (10 µg/L) was continuously introduced through a separate channel to correct for potential signal drift and matrix effects, see Rui et al. (2016) for details. The samples were quantified by calculating the ratio of the intensity of the mass spectral signals of Cd to those of the internal standard, following the established calibration curve. The Cd translocation factor (TF) was calculated based on the Cd content in plant roots and stems: ${\displaystyle \mathrm{TF}\left(\text{\%}\right)=\left(\frac{\mathrm{Cs}}{\mathrm{Cr}}\right)\times 100}$

where TF is the percentage of shoot (Cs) to root (Cr) Cd concentration. This factor provides insights into a plant’s ability to absorb heavy metals from the soil and transport them to above-ground tissues.

Chloroplastidic pigment analysis

The roots and leaves of 10 seedlings in one pot were harvested separately 6 days after Cd treatment as a replicate for analysis of chloroplastidic pigments and other substances below. The extraction, detection and content analysis of chloroplastidic pigments are described in Zayneb et al. (2015). Briefly, 200 mg of fresh leaves were rapidly ground with two mL of pre-cooled 95% ethanol in combination with 0.1% CaCO₃ to prevent chlorophyllase activity. The extract was filtered and diluted to 25 mL with 95% ethanol. Absorbance values were determined at 665 nm, 646 nm and 470 nm using 95% ethanol as a blank.

H₂O₂ and O₂^•− analysis

For H₂O₂ measurement, one mL of pre-cooled acetone was added to 100 mg of liquid nitrogen-ground samples. Then the mixture was centrifuged at 12,000 rpm at 4 °C for 20 min, and the supernatant was collected. The H₂O₂ levels were assessed in accordance with the guidelines provided by the Hydrogen Peroxide Content Assay Kit (Sangon Biotech, #D799774, Shanghai, China). For O₂^•− analysis, one mL of pre-chilled phosphate-buffered saline (pH 7.8) was added to 100 mg of cryogenically ground samples. Following centrifugation under the same conditions, the supernatant was collected. The O₂^•− producing rate in root and leave extracts were evaluated following the protocol outlined in the Superoxide Anion Content Assay Kit (Sangon Biotech, #D799771, Shanghai, China).

SOD and POD analysis

The frozen samples (100 mg) ground in liquid nitrogen were thawed on ice, and one mL of 0.05 M potassium phosphate buffer (PPB, pH 7.8) was added. After homogenization, the mixture was centrifuged at 12,000 rpm at 4 °C for 20 min, and 200 µL of supernatant was collected for SOD activity measurement. The reaction system for the POD activity of one sample determination contained 2.7 mL of 14.5 mM methionine, 0.1 mL of 3mM EDTA-Na₂, 0.1 mL of 60 µM riboflavin, 0.1 mL of 2.25 mM nitro-blue tetrazolium (NBT) and 40 µL of supernatant. After mixing, the tubes were incubated under 4,000 lux at 25 °C for 20 min. Absorbance at 560 nm (OD560) was measured using a SpectraMax M5 microplate spectrophotometer (Molecular Devices, San Jose, CA, USA), with an unilluminated tube as blank control. Commercial SOD (Sigma-Aldrich, St. Louis, MO, USA 100 U/mL) served as positive control, while a sample-free reaction was included as negative control.

The frozen samples (100 mg) ground in liquid nitrogen were thawed on ice, and one mL of 0.1 M potassium phosphate buffer (PPB, pH 7.0) containing 1% polyvinylpyrrolidone (PVP) was added. After homogenization, the mixture was centrifuged at 12,000 rpm at 4 °C for 20 min, and the supernatant was collected for POD activity measurement. For POD activity determination, the reaction mixture consisted of 1.8 mL of 0.1 M HAC buffer (pH 5.4), one mL of 0.75% guaiacol, 50 µL of supernatant, and 0.1 mL of 3% H₂O₂. After mixing, absorbance at 460 nm was recorded every 15 s for 3 min.

NPSH analysis

The extraction and content analysis of non-protein sulfhydryl compounds (NPSH) were performed according to Morelli & Scarano (2004) with a slight modification. Briefly, roots or leaves (about 500 mg each) were ground in liquid nitrogen and one mL of extraction solution containing 5% (w/v) sulfosalicylic acid and 6.3 mM diethylenetriaminepentaacetic acid (DTPA) was added to each sample. The mixture was centrifuged (12,000 rpm) at 4 °C for 15 min, and the supernatants were collected. The reaction system for NPSH analysis contained 2.1 mL of 0.5 M K₂HPO₄, 85 µL of 10 mM 5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB) and one mL of supernatant. After reacting at 30 °C for 2 min, absorbance at 412 nm was measured at 412 nm. Results were plotted against a standard curve generated from one mM reduced GSH.

Proline analysis

Roots or leaves (approximately 100 mg each) were homogenized in liquid nitrogen, followed by the addition of one mL of a 3% (w/v) sulfosalicylic acid solution to each sample. The samples were then subjected to boiling water for 10 min before centrifugation at 8,000 rpm for 15 min, after which the supernatant was carefully collected. To this supernatant, 400 µL of glacial acetic acid and 400 µL of acidic ninhydrin were added and reacted in boiling water for an additional 30 min. After cooling, 600 µL of toluene was added and the mixture was vortexed for 30 s. The upper phase was collected and centrifuged at 3,000 rpm for 5 min (Nayyar & Walia, 2003). Absorbance was subsequently measured at a wavelength of 520 nm using toluene as a blank. Proline content was determined using a standard curve (0–100 µM) prepared from 1 mM L-proline standard (Sigma-Aldrich, St. Louis, MO, USA ≥99% purity), with each concentration assayed in triplicate.

Data analysis

The data were analyzed using Statistica 6. Results of all the bar graphs are the average of three independent determinations + SE. Differences in the levels of Cd and endogenous substances were analyzed by Student’s t test. Differences in germination rate and germination potential among different concentrations of Cd were analyzed by one-way ANOVAs (P < 0.05; Duncan’s multiple range test).

Cd treatment inhibited seed germination

To explore if Cd exposure affect the seed germination of okra, we conducted the experiments on GR and GP with Cd at different concentrations ranging from 0.5 to 2 mM. Exogenous application of Cd inhibited GR and GP of okra in a dose-dependent manner (Figs. 1C, 1D). For red okra, all concentrations significantly reduced GR by the 7th day (Figs. 1A, 1B). Similarly, in green okra, all concentrations except 0.5 mM significantly inhibited seed germination (Figs. 1A, 1B). Notably, at two mM Cd exposure, the germination rates were markedly reduced to 53% (95% CI and p < 0.001) for red okra and 48% (95% CI and p < 0.001) for green okra.

The Cd accumulation in okra plant

After subjecting both red and green okra seedlings to a 6-day treatment with 50 µM Cd, significant Cd accumulation was observed in the shoots and roots of both okra varieties. In red okra, the Cd content peaked at 287.8 mg kg⁻¹ DW in the shoots and 790.3 mg kg⁻¹ DW in the roots (Fig. 2A). In green okra, the Cd content reached 280.8 mg kg⁻¹ DW in the shoots and 903.7 mg kg⁻¹ DW in the roots (Fig. 2B). Calculations revealed that the translocation factor (TF) of Cd stood at 49.24% in red okra and 30.73% in green okra. The TF metric signifies the percentage of Cd effectively translocated from the roots to the shoots within the plant system.

Cd treatment inhibited plant growth

To explore the effect of Cd on plant growth, we conducted the experiment adding 50 µM Cd to the nutrient solution. As shown in Fig. 2, Cd treatment significantly inhibited the growth of both red okra (Fig. 3A) and green okra (Fig. 3B). The length of shoots of red and green okra decreased by 20.3% (95% CI and p = 0.02) and 25.9% (95% CI and p = 0.0013) (Fig. 4A, Left), respectively, while the length of their roots decreased by 45.3% (95% CI and p < 0.001) and 27.0% (95% CI and p < 0.001) (Fig. 4A, Right). We also tested the dry and fresh weight of okra. Compared with the controls, both varieties exhibited a significant reduction in fresh weight (Fig. 4B) and dry weight (Fig. 4C). The moisture content of okra plants is almost unaffected by Cd treatment, with only the roots of Cd-treated green okra being 1.9% (95% CI and p = 0.045) lower than the control (Fig. 4D).

Cd treatment increased chloroplastidic pigment content

The impact of Cd treatment on chloroplastidic pigment content in both red okra and green okra was explored. The results demonstrated that Cd treatment elicited distinct responses in the levels of chloroplastidic pigments in two plant varieties studied. Specifically, in red okra, Cd application resulted in a significant 14.4% increase (95% CI and p = 0.011) in chlorophyll a content and a 32.4% increase (95% CI and p = 0.05) in carotenoid content, whereas no significant effect was observed on chlorophyll b level (Fig. 5A). However, in green okra, Cd treatment led to a significant increase in the levels of all three pigments: chlorophyll a (37.4%, 95% CI and p = 0.002), chlorophyll b (46.4%, 95% CI and p < 0.001), and carotenoids (42.1%, 95% CI and p = 0.006) (Fig. 5B). Also, the increase in the chloroplastidic pigments was greater in green okra than in red okra.

Cd treatment increased H₂O₂ and O₂^•− production

We examined changes in H₂O₂ and O₂^•− levels under Cd stress. Treatment with 50 µM Cd significantly increased H₂O₂ levels in the roots of both red and green okra, with red okra exhibiting a 2.23-fold (95% CI and p = 0.03) increase and green okra a 1.4-fold (95% CI and p = 0.035) increase compared to controls (Fig. 6B), but not in their leaves (Fig. 6A). It is worth noting that red okra leaves contain higher levels of H₂O₂ than green okra. After 6 days of Cd treatment, O₂^•− production showed significantly increased level compared to control, O₂^•− production in leaves rose by 182.3% (95% CI and p < 0.001) in red okra and 120.9% (95% CI and p = 0.085) in green okra, while root activity surged by 298.8% (95% CI and p < 0.001) in red okra and 505.8% (95% CI and p < 0.001) in green okra (p < 0.05, 95%, Fig. 6D). Notably, red okra leaves exhibited higher O₂^•− production than green okra under the same Cd concentration (Fig. 6C).

Cd treatment increased the activity of antioxidant enzymes

In Cd-treated red and green okra, both POD and SOD showed significantly increased activity compared to controls. POD activity in leaves rose by 58.2% (95% CI and p = 0.001) in red okra and 233.7% (95% CI and p < 0.001) in green okra (Fig. 7A), while root activity surged by 959.3% (95% CI and p = 0.003) in red okra and 1,461% (95% CI and p < 0.001) in green okra (Fig. 7B). Similarly, SOD activity in roots increased by 229.3% (95% CI and p = 0.021) in red okra and 457.8% (95% CI and p < 0.001) in green okra (Fig. 7D). In contrast, leaf SOD activity showed only a modest 1.60-fold (95% CI and p = 0.029) increase in green okra, with minimal change in red okra (Fig. 7C). These results highlight a stronger Cd-induced antioxidant response in roots compared to leaves.

Cd treatment increased proline content

In red okra, proline content increased significantly only in the roots, reaching 3.09 (95% CI and p < 0.001) times higher than the control (Fig. 8B). In contrast, green okra showed elevated proline in both roots and leaves, with levels 8.45 (95% CI and p < 0.001) and 6.11 (95% CI and p = 0.003) times higher than controls, respectively (Figs. 8A, 8B). These results highlight varied proline modulation under Cd stress between the two varieties.

Cd treatment increased NPSH levels

After 6 days of Cd treatment, both red and green okra showed significant NPSH induction in the roots, with red okra exhibiting a 13.52-fold (95% CI and p = 0.006) increase and green okra a 10.21-fold (95% CI and p < 0.001) increase compared to controls (Fig. 9B). In contrast, leaf NPSH levels were less affected, with red okra showing a 1.25-fold (95% CI and p = 0.028)) increase and green okra a 1.82-fold (95% CI and p < 0.001) increase (Fig. 9A). These results indicate a stronger Cd-induced NPSH response in roots than in leaves.