Plant growth parameter (growth rate, seedling length, root length, and shoot length)
Increased industrialization and urbanization have markedly contributed to soil contamination by Cd which impairs plant growth and development, as well as reduction of plant metabolism (de Souza Costa et al. 2012). It is established that plant accessions vary in their tolerance to Cd toxicity. Therefore in this study, two contrasting wild castor accessions were used to investigate the growth, biomass production, TI, antioxidant system, and lipid peroxidation in castor under Cd stress for 5 days. The results showed a wide variation between the accessions with respect to all the parameters investigated (Table 1, Figs. 1 and 2). The growth rate of the castor accessions were inhibited upon Cd treatment in the growth medium compared to their control groups and symptoms of Cd toxicity such as root darkening occurred. The inhibitory effect of Cd was more severe in S2-4 compared to 16–024. Mostly low Cd exposure (below 5 µM) results in low or no phytotoxicity depending on the plant species (Yang et al. 2018). However high Cd concentration, especially above 100 mg/kg has been associated with massive reduction in plant growth accompanied with high phytotoxicity such as leaf spot, chlorosis, and necrosis (Shi and Cai 2009; Yang et al. 2018). Hence, our results of Cd toxicity and reduction in growth rate observed in the castor accessions compared to the control may also be due to the high Cd concentration which restricted the intake of essential nutrients in the growth medium thereby disrupting the plant metabolism. These findings are in consonance with previous studies reported in kenaf (Li et al. 2013), maize (Xu et al. 2014), castor (Hazama et al. 2015), sugarcane (Zeng et al. 2017), and switchgrass (Guo et al. 2019). This present study confirms that high Cd concentration causes cytotoxicity and reduction in plant growth.
The seedling height, root length, and shoot length of the castor accessions in Cd stress decreased compared to their controls (Table 1). Cd stress decreased seedling height of plants by 25.8 % in 16–024 and 43.8 % in S2-4 with respect to their control group. Compared to the control, the decline in root and shoot length of 16–024 were 27.7 % and 24.3 %, respectively; the decreased values of root and shoot length of S2-4 were 50.0 % and 36.6 %, suggesting a higher decrease in S2-4 than 16–024, respectively. A possible explanation for the higher decrease in height, root and shoot length in S2-4 may be due to its higher sensitivity to Cd stress. Also, a similar study has reported a wide variation between two kenaf accessions when exposed to 20 µ/mol Cd in the growth medium. It was found that the kenaf Cd-sensitive cultivar ‘ZM412’ recorded a higher decrease in root and shoot length compared to the Cd-tolerant cultivar ‘Fuhong 991’ (Li et al. 2013). Yang et al. (2018) reported a significant reduction in length which varied among seven genotypic Salix species upon Cd exposure.
Biomass response under Cd stress
Biomass reduction is a general response of higher plants and an important indicator to evaluate metal toxicity in plants. Previous studies have reported a decline in root and shoot biomass due to Cd stress (de Souza Costa et al. 2012; Shi and Cai 2009; Xu et al. 2014; Zeng et al. 2017). Table 1 shows that the fwt and dwt of biomass of the castor accession under Cd stress decreased compared to their control groups. Suppression of fwt of root and shoot biomass in 16–024 were 47.1 % and 59.2 %, respectively, and that of S2-4 were 64.8 % and 65.3 %, compared to their control groups. Compared to control group, the root and shoot dwt of 16–024 significantly decreased (p < 0.05) and insignificantly increased (p > 0.05), respectively, by 11.5 % and 2 %. The insignificant increase in shoot dwt of 16–024 suggests certain level of tolerance in this accession to Cd stress. Conversely, S2-4 had both root and shoot dwt decreased (p > 0.05) by 36.0 % and 21.7 %, respectively compared to control groups (Table 1). The biomass variation between the two castor accessions in response to Cd stress could be attributed to the differences in their genetic makeup. 16–024 was relatively tolerant to Cd stress and exhibited a minimal reduction in all growth parameters and biomass production compared to S2-4 in the growth medium. Also, it was observed that, except shoot dwt, all the other biomass parameters (Table 1) tested decreased, and hence this proposes that Cd toxicity has varied effect among the plant parts. Nevertheless, to better understand and distinguish the tolerance ability between the two castor accessions, the TI and biochemical mechanisms were further investigated in this study.
Table 1
Growth and biomass of two castor accessions under control and Cd treatment
Cd treatment (mg/L)
|
Seedling height (cm)
|
Root length (cm)
|
Shoot length (cm)
|
Fresh weight of biomass (g/plant)
|
Dry weight of biomass (g/plant)
|
Root
|
Shoot
|
Root
|
Shoot
|
16–024
|
|
|
|
|
|
|
|
0
|
36.2 ± 2.13
|
16.0 ± 1.00
|
20.2 ±
1.13
|
3.18 ±
0.12
|
8.61 ± 0.15
|
0.09 ±
0.01
|
0.15 ±
0.01
|
100
|
26.8 ± 1.23*
|
11.6 ± 0.54*
|
15.3 ±
0.70*
|
1.68 ±
0.08*
|
3.51 ±
0.03*
|
0.08 ±
0.01*
|
0.15 ±
0.02ns
|
t value
|
17.925
|
16.513
|
19.324
|
24.459
|
73.612
|
17.321
|
-0.309
|
|
(p = 0.003)
|
(p = 0.004)
|
(p = 0.003)
|
(p = 0.002)
|
(p = 0.001)
|
(p = 0.003)
|
(p = 0.787)
|
S2-4
|
|
|
|
|
|
|
|
0
|
36.2 ±
1.43
|
19.3 ±
0.76
|
16.8 ±
0.67
|
2.50 ±
0.04
|
5.45 ±
0.13
|
0.08 ±
0.01
|
0.15 ±
0.00
|
100
|
20.3 ±
1.11*
|
9.7 ±
0.54*
|
10.7 ±
0.57*
|
0.88 ±
0.02*
|
1.89 ±
0.02*
|
0.05 ±
0.01 ns
|
0.12 ±
0.02 ns
|
t value
|
85.354
|
75.432
|
106.63
|
69.286
|
43.71
|
3.928
|
3.20
|
|
(p = 0.001)
|
(p = 0.001)
|
(p = 0.001)
|
(p = 0.001)
|
(p = 0.001)
|
(p = 0.059)
|
(p = 0.085)
|
Data are mean of three replicates ± standard deviation. Paired-Samples T-test (0.05) were performed to compare the means of control and treatment, where * = significant and ns = not significant.
Tolerance index
Tolerance index is a useful indicator to assess plant tolerance under metal stress. Indexes with high value signify higher tolerance and indexes with low value indicates lower tolerance or higher metal effect on plant (Shi and Cai 2009). The TI of seedling height, root length, shoot length, root dwt, shoot dwt, root fwt, and shoot fwt are shown in Fig. 1. Comparatively, the TI of seedling height of 16–024 significantly increased (p < 0.05) by 74.2 % and S2-4 by 56.2 % (Fig. 1.a). Also, the TI of root and shoot length of 16–024 were 72.3 % and 75.7 %, respectively. Those values were significantly higher than the root length (p < 0.05) and shoot length (p < 0.05) of S2-4 which increased by 50.0 % and 63.3 %, respectively. For root and shoot fwt 16–024 significantly increased by 52.9 % and 40.8 % respectively, compared to the root fwt and shoot fwt of S2-4 which were 35.2 % and 34.7 %. The TI of root dwt of 16–024 was higher (88.4 %) but not significant (p > 0.05) compared to the root dwt of S2-4 (64.8 %). Similarly, the paired-samples T-test showed that there was no significant difference (p > 0.05) between the shoot dwt of 16–024 (101.9 %) and S2-4 (78.4 %). The higher tolerance of 16–024 under Cd stress is attributed to the higher values compared to S2-4. Recently, Palanivel et al. (2020) reported high TI of castor plant height (48–133 %), root dry wt. (25–360 %), and shoots dry wt. (18–328 %) when grown in five different types of copper mined soils and slag. The high TI value reported in their study compared to this present study may be associated with differences in metals and experimental conditions.
Antioxidative enzymatic activities
To combat Cd-induced oxidative damage, the plant’s cell and organelle employ different antioxidant enzymes that help to control redox potential and tolerate oxidative stress. The production of antioxidant enzymes including SOD, CAT, among others play important roles to sequester, neutralize or detoxify Cd toxicity as a result of ROS production, thereby enhancing plant tolerance (Singh et al. 2016; Yeboah et al. 2020a). SOD is a metalloprotein that catalyzes the dismutation of two molecules of O2− to O2 and H2O2 and helps protect cells against the toxic effects of O2− produced in different cellular components (Bhaduri and Fulekar 2012) and CAT is a primary enzyme that regulates intracellular H2O2 level and catalyzes into H2O and divalent oxygen (Bhaduri and Fulekar 2012). The activities of SOD and CAT in the two castor accessions under Cd stress and their control groups are shown in Fig. 2a and b. The activity of CAT significantly decreased (p < 0.001) in 16–024 and increased (p < 0.05) in S2-4 compared to their control group. SOD and its activity significantly increased (p < 0.001) in the castor accessions under Cd treatment compared to their control groups. Increases in SOD and CAT activities were higher in S2-4 (1002.0 ± 12.20 U/g fw and 18.97 ± 2.40 U/g fw) than 16–024 (891.9 ± 11.47 U/g fw and 5.30 ± 0.32 U/g fw). The higher SOD and CAT activities in S2-4 may be explained by the higher production of ROS in the leaves which was effectively converted to H2O2 by SOD and further converted to reusable by-products (water and oxygen molecules) by CAT. On the other hand, the decrease in CAT activity in 16–024 may be due to high reactive singlet molecular oxygen which inhibited the enzyme activity or disruption of protein synthesis caused by Cd toxicity (Ma et al. 2017). Another possible reason for the decrease in CAT may be due to the fact that the enhanced levels of SOD could not be sufficient to remove completely the generated ROS, which are capable of provoking the inhibition of CAT. The better coordination of enzymatic activities as witnessed by the maximum (S2-4) increases in SOD and CAT in the leaves of the S2-4 castor accession suggest effectiveness as the first line of defense in scavenging ROS and function in Cd detoxification in the leaves of castor plant. In agreement with our study, Zeng et al. (2 017) reported elevated levels of SOD activities (30.31-150.15 %) in the leaves of sugarcane among five different accessions which effectively eliminated oxidative stress caused by Cd toxicity. Existing studies have also reported enhanced SOD activities upon Cd stress (He et al. 2013; Li et al. 2013) except for Xu et al. (2014) who reported a decline SOD activity in maize seedlings which was attributed to inhibition caused by increasing H2O2 in the Cd treatment. Similarly, increase and decrease in CAT activity have been reported in rice (Yu et al. 2013), poplar (He et al. 2013), castor (Zhang et al. 2015), and sugarcane (Zeng et al. 2017) upon Cd exposure in the growth medium, which can be attributed to the different plant species and tissues, or concentrations and durations of metal exposure (Zhang et al. 2015). The different responses of antioxidant enzymes (SOD and CAT) in our study to Cd seem to be genotype-specific.
Non-antioxidant enzymes
GSH is a key component of non-protein thiols responsible for maintaining the cellular redox status. GSH acts as a chelating bio-ligand that neutralizes the oxidative stress caused by metal toxicity in plants. It also plays an important role by scavenging excess H2O2 and reacts with hydrogen radical, superoxide radical, and singlet oxygen (Cobbett 2000; Hall 2002). Increasing GSH levels is important for Cd detoxification since it is a precursor of PCs (components of non-protein thiols), which form complexes with Cd and sequester it into the vacuoles (Cobbett 2000). However, the present results revealed a concomitant decrease of GSH level in both accessions (Fig. 2c), with the effect being more severe in S2-4 (3.54 ± 0.24 µmol/g) than 16–024 (2.0 ± 0.14 µmol/g) which may be due to the increase of PC leading to higher resistance of plants to oxidative stress (Zhang et al. 2014). This finding conforms with that reported in castor (Zhang et al. 2014), Brassica chinensis L (Lou et al. 2017), and Zea mays L. (Singh et al. 2019), where decreased GSH levels under Cd stress were associated with the PC synthesis. Thus, it may be deduced that the depletion of GSH to reduce the toxicity of Cd could be a primary mechanism for plants to adapt to Cd stress.
Lipid peroxidation
MDA is the cytotoxic product of membrane lipids peroxidation, and it accumulates when plants are exposed to oxidative stress. Therefore, the MDA content is mostly considered as a basic biomarker of lipid peroxidation and the stress level (Guo et al. 2014). In this study, we found that accumulation of MDA decreased (p > 0.05) by 0.44 ± 0.15 nmol/g in accession 16–024 and significantly increased (p < 0.05) by 2.67 ± 0.07 nmol/g in accession S2-4, upon exposure to Cd as compared to their controls (Fig. 2d). This result is consistent with that reported in two different rice accessions, where MDA content decreased in Cd-tolerant cultivar ‘Jaya’ and increased in Cd-sensitive cultivar ‘Ratna’ when treated with Cd (100 and 500 µM) stress for 20 days in the growth medium (Shah et al. 2001). Bauddh and Singh (2016) also reported higher MDA contents in the leaves (4.53 fold ) of Brassica juncea L. compared to the leaves (2.8 fold) of castor when subjected to Cd treatment. MDA levels reflect the degree of cell membrane damage caused by oxidative stress. Hence, the increased MDA content of Cd-sensitive accession S2-4 indirectly explains the oxidative stress induced by Cd stress compared with that of 16–024. The decreased MDA content in 16–024 may be explained by the quick removal of ROS by antioxidant enzymes particularly, SOD and CAT which acts as the first line of defense against free radicals. Higher SOD activities in different cellular compartments under Cd stress signifies higher O2−, capable of causing oxidative damage (Zhang et al. 2015). Therefore, the growth and decrease in MDA content in the leaves of 16–024 may be attributed to the low accumulation of free radicals which was effectively removed by SOD. This result may explain the higher tolerance to Cd stress in 16–024 compared to S2-4.