Root tolerance and biochemical response of Chinese lettuce (Lactuca sativa L.) genotypes to cadmium stress

This study was conducted to determine the root tolerance and biochemical responses of four Chinese Lactuca sativa L. genotypes (Lüsu, Lümeng, Yidali and Anyan) to cadmium (Cd2+) stress. Twenty-eight days old seedlings were exposed to Hoagland’s nutrient solution supplied with or without 100 µM CdCl2 and monitored for seven days in a climate controlled room. The 100 µM CdCl2 significantly (P < 0.001) decreased all the root morphological indexes of the four genotypes. However, Yidali, which possessed the smallest root system, exhibited greater root tolerance to Cd2+ by having the highest tolerance indexes for root volume (46%), surface area (61%), projected area (74%) and numbers of root forks (63%) and root tips (58%). Moreover, Cd2+ stress also caused increases in H2O2 contents in the roots but the increase was least in Yidali which showed greater root tolerance to Cd2+stress. The effect of Cd2+ stress on the contents of hormones in the roots depended on the genotypes. Under Cd2+ stress, abscisic acid correlated positively with indole-3-acetic acid (r = 0.669*), gibberellic acid (r = 0.630*) and cytokinin (r = 0.785**). The antioxidant enzyme activities and proline responses of the four genotypes to Cd2+ stress were similar. The SOD activity was decreased whiles the CAT and POD activities, as well as the contents of proline increased in all the genotypes under the stress condition. These results suggest that lettuce genotypes with smaller root systems could be more tolerant to Cd2+ stress compared to those with larger root systems.


INTRODUCTION
Heavy metals are a major cause of abiotic stress in plants and their accumulation in human and animal tissues leads to serious health problems (Singh et al., in press). The oxidative stress caused by heavy metals including cadmium (Cd 2+ ) results in membrane damage and also decreases photosynthesis (Su et al., 2017;Kaur et al., 2017). Increases in the levels of reactive oxygen species (ROS) such as hydrogen peroxide (H 2 O 2 ), superoxide anion and thiobarbituric acid reactive substances (TBARS) in plant cells is the main cause of oxidative stress in plants (Tripathi et al., 2017). Cd 2+ toxicity increases the accumulation of H 2 O 2 , superoxide anion and TBARS in lettuce (Wang et al., 2019) and in Arabidopsis thaliana (Song et al., 2017). The effect of Cd 2+ on root morphology varies with different plant species (Li et al., 2009;Lu et al., 2013). Lambrechts et al. (2013) found that the root diameter of Lolium perenne and Trifolium repens increases in relation to a high number of short and wide adventitious roots under Cd 2+ stress. Moreover, in Zea mays (Maksimovi'c et al., 2007) and in Salix alba (Lunáčková et al., 2004) plants, the root diameters increases under Cd 2+ stress. The root length of lettuce (Lactuca sativa L.) plants exposed to Cd 2+ decreased by 89% (Bautista, Fischer & Cárdenas, 2013). In A. thaliana, Cd 2+ stress decreased root length but doubled the diameter of the lateral roots (Vitti et al., 2013). In view of the variations in the response of plants root systems to Cd 2+ stress, Lu et al. (2013) indicated that the response of plants root systems to Cd 2+ stress can be used in assessing their tolerance to Cd 2+ stress.
Plant hormones are a group of chemical messengers that are primarily known to regulate plant growth and development (Peto' et al., 2011) but they also play roles in plants tolerance to biotic and abiotic stresses (Krishnamurthy & Rathinasabapathi, 2013). Christmann et al. (2006) reported that increases in endogenous abscisic acid (ABA) contents in plants improve their tolerance to various stresses. The endogenous levels of ABA in rice (Kim et al., 2014) and potato tubers (Stroinski et al., 2010) as well as in the roots of two aquatic plants, Typha latifolia and Phragmites australis (Fediuc, Lips & Erdei, 2005) increased under Cd 2+ stress. Veselov et al. (2003) also found that the contents of cytokinin in the shoots and roots of wheat decreased within 1-2 h of exposure to Cd 2+ . Auxins, including indole-3-acetic acid (IAA) also play important roles in protecting and regulating plants metabolism under stress conditions. Generally, heavy metal stress decreases the levels of endogenous auxins (Gangwar et al., 2014).
Plants tolerance to heavy metals has been associated with increase activities of antioxidant enzymes (Yang & Ye, 2015). However, Fodor (2002) reported that high Cd 2+ concentrations resulted in decreased antioxidant activities in plants. The catalase (CAT) activity in several plants including, Lactuca sativa (Wang et al., 2019;Monteiro et al., 2009), Phaseolus vulgaris (Chaoui et al., 1997 and Pisum sativum (Chaoui & El Ferjani, 2005) decreased when the plants were subjected to Cd 2+ stress. The CAT activity in pea seedlings was also decreased whiles the superoxide dismutase (SOD) and peroxidase (POD) activities increased under Cd 2+ stress (Agrawal & Mishra, 2007). The rates of POD activity in the roots and shoots of pea seedlings were higher at lower Cd 2+ (5-20 µM CdCl 2 ) concentrations than at higher Cd 2+ (50-100 µM CdCl 2 ) concentrations (Bavi, Kholdebarin & Morashahi, 2011). Proline, which is a component of the non-specific defense systems against heavy metal toxicity acts as a chelator as well as a protein stabilizer in plants (Sharma & Dubey, 2005). The contents of proline increased in lettuce plants under Cd 2+ stress (Jibril et al., 2017) and in wheat plants exposed to Pb stress (Lamhamdi et al., 2013).
Different plant species possess a wide range of protection mechanisms against Cd 2+induced stress (Mohamed et al., 2012). However, the response of plants to Cd 2+ stress depend on several factors including the genotype and the Cd 2+ concentration of the growing medium (Irfan, Ahmad & Hayat, 2013). The root system of plants is the foremost organ that comes into contact with Cd 2+ in a growing medium. Therefore, the response of the root system to Cd 2+ stress could have greater impact on the tolerance of the whole plant to the stress. Richard et al. (2015) screened maize lines for Al tolerance and indicated that root tolerance should be considered in selecting cultivars for Al tolerance. In Brassica juncea seedlings exposed to Cd 2+ stress, the POD and CAT activities of the root system were more efficient in scavenging ROS compared with the shoot system (Mohamed et al., 2012). Although a number of studies have reported the effect of Cd 2+ on lettuce plants (Wang et al., 2019;Bautista, Fischer & Cárdenas, 2013;Monteiro et al., 2009;Monteiro et al., 2007), the root tolerance and biochemical response of the root system of different lettuce genotypes to Cd 2+ stress have rarely been reported. Moreover, to the best of our knowledge, the response of the genotypes studied in the present experiment to Cd 2+ stress has not been reported.
The objectives of our study were: (1) to evaluate the root tolerance of four lettuce genotypes to Cd 2+ stress based on root tolerance indexes, (2) to determine the biochemical (hormones, antioxidant enzymes activities and proline) defense response of the four lettuce genotypes to Cd 2+ stress.

Genotypes used for the experiment
The experiment was conducted at the College of Horticulture, Gansu Agricultural University, Lanzhou, Gansu Province, PR China (36 • 03 N, 103 • 40 E). The seeds of four improved lettuce (Lactuca sativa L.) genotypes obtained from the Gansu Academy of Agricultural Sciences, Lanzhou, China, were used in this experiment (Table 1).

Seedling growth conditions
The seeds were germinated in petri dishes within 48 h under light condition in a climate box (temperature 20 • C; light intensity 200 µmol m −2 s −1 photosynthetically active radiations, PAR; relative humidity 80%). The germinated seeds were sown in trays which were filled with vermiculite and the seedlings were raised in a greenhouse (average daily temperature 24 ± 2 • C; relative humidity 60-70%; 12 h light) for two weeks. During the first two weeks of growth, the young seedlings were irrigated with half-strength Hoagland's nutrient solution with pH adjusted to 6.0 (Hoagland & Arnon, 1950). After the first two weeks of growth, the seedlings were grown hydroponically for another two weeks using full-strength Hoagland's nutrient solution in a climate controlled room (25 • C/22 • C light/dark periods, 75% relative humidity and 14 h light period with 200 µmol m −2 s −1 PAR). In all, 240 seedlings were transferred to 24 opaque hydroponic containers (length 82 cm; width 30 cm; height 5 cm). Each of the containers were filled with 5 L of the full-strength Hoagland's nutrient solution and supplied with 10 seedlings of a particular lettuce genotype. The nutrient solution was changed at 4 days interval until the seedlings were four weeks old before the 100 µM CdCl 2 treatment was applied according to the experimental design.

Experimental design and treatments
A 4 × 2 factorial experiment in a completely randomized design with 3 replications was conducted in the climate controlled room using 28 days old seedlings. The treatments consisted of the four lettuce genotypes and two Hoagland's nutrient solutions. Two hundred and forty seedlings (60 seedlings per genotype) were grouped into two. Half of the seedlings of each genotype were exposed to the Hoagland's nutrient solution supplemented with 100 µM CdCl 2 and the other half were maintained in the nutrient solution without 100 µM CdCl 2 , as control plants. We used the 100 µM CdCl 2 concentration in our experiment based on previous studies that were conducted with lettuce as the test crop (Xu et al., 2014;Monteiro et al., 2009;Monteiro et al., 2007). At seven days after exposure to treatments, one plant each was taken randomly from each experimental unit and used for the analysis of root morphological indexes. At the same time, five plants were also taken randomly from each experimental unit and their root tissues were immediately stored at −80 • C for biochemical analysis.

Root morphological indexes
The roots of the sampled plants were gently separated from the shoots, rinsed in distilled water and the root systems were scanned with the aid of a root scanner (STD 4800, EPSON, Canada) to obtain the digital images. The total root length (TRL), root volume (RV), root surface area (RSA), root projected area (RPA), and numbers of root tips (NRT) and root forks (NRF) per plant were determined using the root image analysis software, Win RHIZO version 5.0 (Regent Instruments, Inc., Quebec City, Canada).

Determination of root tolerance indexes
The Cd 2+ tolerance indexes (TI) of the plants root systems which indicates the resistance of the root systems to the heavy metal stress was calculated as described by Wilkins (1978) with some modifications. Since the various root morphological indexes such as total root length, root volume, number of root tips and root surface area have direct influence on Cd 2+ uptake and the plants tolerance to Cd 2+ stress, we calculated the Cd 2+ TI for each of the root indexes at the end of the experiment. Based on the TI score, we considered the genotype which had the highest TI for most of the indexes measured as the tolerant genotype among the genotypes we evaluated. The TIs were calculated as follows: TI = Root index under Cd stress Root index without Cd stress × 100.

Determination of H 2 O 2 contents in root samples
Contents of H 2 O 2 in the root samples were determined following the procedure described by Junglee et al. (2014) but with slight modifications. Briefly, we 0.1 g fresh root sample was ground with liquid nitrogen in mortar with pestle and the homogenate transferred into a 2 mL capacity centrifuge tube which was then kept in ice bath. We added 1.5 mL 0.1% TCA and the homogenate were centrifuged at 12,000× g for 15 min under 4 • C. Then, 0.5 ml of the supernatant was thoroughly mixed with 0.5 mL PBS (pH 7.0) and 1 mL 1 mol L −1 KI. The mixture was placed under constant temperature (28 • C) for 1 h. The absorbance was measured at 390 nm and the contents of H 2 O 2 were calculated using the H 2 O 2 reference standard curve (0, 1, 2, 3, 4 and 5 mmol L −1 ).

Determination of contents of hormones in root samples
Fresh root samples (0.5 g) of each of the four lettuce genotypes were collected from three plants in each experimental unit, quickly wrapped in alumnum foil, labeled and then put in liquid nitrogen. After this, the samples were put in labeled plastic bags and stored in a freezer at −80 • C. The contents of ABA, IAA, GA 3 and Cytokinin were later determined by the Enzyme Linked Immunosorbent Assay (ELISA) technique (Shanghai Jiwei Biological Technology Co. Ltd., PR China).

Antioxidant enzymes activities and proline contents in roots
Enzymes assays were done using frozen root tissues that were stored at −80 • C. For each enzyme, about 0.5 g of the root tissue was ground in liquid nitrogen with a mortar and pestle and then homogenized in 5 ml of 0.1 M phosphate buffer (pH 7.5), containing 0.5 mM ethylene-diamine-tetra-acetic acid (EDTA). Each homogenate was centrifuged at 12,000× g for 15 min at 4 • C, and the supernatant was aliquoted for determination of enzymatic activity; superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6) and peroxidase (POD, EC 1.11.1.7) activity assays. The SOD activity was measured by determining the ability of plants to inhibit the photochemical reduction of nitroblue tetrazolium (NBT). Based on the inhibition of 50% of NBT photoreduction as one unit of SOD activity, the amount of reduced NBT was monitored spectrophotometry-colorimetry at 560 nm (Giannopolitis & Ries, 1977). The POD activity was estimated at 470 nm as described by Chance & Maehly (1955). The CAT activity was measured by determining the oxidation of H 2 O 2 and monitored as a decline spectrophotometry-colorimetry at 240 nm (Nakano & Asada, 1981). Proline contents in the root samples were determined after extraction at room temperature with 3% of 5-sulfosalicylic acid solution as previously described (Bates, Waldren & Teare, 1973).

Data analysis
The data were subjected to analysis of variance and the treatment means were separated by the least significance difference (LSD) test at 5% using Genstat statistical software (9th edition; Lawes Agricultural Trust, Rothamsted Experimental Station, UK). In the figures, the spread of the mean values were shown using the standard deviations. Different letters assigned to the bars indicate significant differences among the treatments by LSD (P < 0.05). Pearson's correlation analysis was used to determine the relationship between root morphological indexes and contents of root hormones under no stress and under Cd 2+ stress conditions. The correlation analyses were done using the average values for every trait × genotype × treatment. Graphs were prepared using Graphpad Prism version 8.0.

Root morphological characteristics
The results of our experiment showed that there was significant (P < 0.001) genotype × Cd 2+ interaction effect on total root length (TRL), root volume (RV), root surface area (RSA), root projected area (RPA), number of root forks (NRF) and number of root tips (NRT) of the lettuce genotypes (Fig. 1). Generally, Cd 2+ stress decreased all the root morphological indexes across the genotypes but Yidali was the least affected. The decrease in TRL due to Cd 2+ was least (39.0%) in Lüsu and greatest (56.0%) in Yidali. The least (38.7%) decrease in RSA was observed in Yidali and the greatest (65.9%) in Lüsu. Moreover, the least (25.6%) and the greatest (59.1%) decreases in RPA were observed in Yidali and Anyan genotypes, respectively. The least (54.0%) decrease in RV also occurred in Yidali whiles the greatest (80.3%) was observed in Lümeng. Cd 2+ stress also decreased NRT and NRF by 42.3%-67.9% and 36.0%-56.8%, respectively in the genotypes and in each case, Yidali was the least affected. The decreases in the root morphological indexes due to Cd 2+ stress resulted in reductions in the sizes of the root systems of the four lettuce genotypes (Fig. 2). Figure 3 shows the results of the root tolerance of the four genotypes to Cd 2+ stress. There was significant (P < 0.001) variations in the tolerance indexes (TIs) among the four lettuce genotypes. The result showed that at seven days after plants exposure to Cd 2+ , Yidali, had the highest TI for most of the indexes measured including RV (46%), RSA (61%), RPA (84%), NRF (65%) and NRT (58%). Based on this result, we considered the Yidali genotype as the most tolerant genotype among the genotypes evaluated. With the exception of TI for TRL which was highest (61%) in Lüsu , the other genotypes had lower TI scores compared with Yidali and these genotypes were considered as sensitive to Cd 2+ stress.

Contents of H 2 O 2 in roots
The results showed significant (P < 0.001) genotype × Cd 2+ interaction effect on contents of H 2 O 2 in the roots of the lettuce genotypes (Fig. 4)

Contents of root hormones
The results showed significant (P < 0.001) genotype × Cd 2+ interaction effect on contents of abscisic acid (ABA), cytokinin, gibberellic acid (GA 3 ) and indole-3-acitic acid (IAA) in the roots of the four lettuce genotypes (Fig. 5)     However, the correlation between cytokinin and RPA was negative (r = −0.579*). The correlation between GA 3 and IAA was positive (r = 0.765**) whiles that between ABA and cytokinin was also positive (r = 0.764**) under Cd 2+ -free condition. The results also showed positive and negative correlations between the hormones and root morphological indexes under Cd 2+ stress condition (Table 2,

Antioxidant enzymes activities and proline contents
The results also showed significant (P < 0.001) genotype × Cd 2+ interaction effect on CAT, SOD and POD activities and contents of proline in the roots of the four lettuce genotypes (Fig. 6). In comparison with their respective control plants, Cd 2+ stress decreased the SOD activities in the roots of all the genotypes by 20.4-40.3%. However, the CAT and POD activities as well as the contents of proline in all the genotypes increased by 16.7-56.6%, 18.6-69.8% and 36.7-79.8%, respectively. The greatest increase in CAT activity was observed in the Yidali genotype whiles the Anyan genotype had the highest POD activity and proline content.

DISCUSSION
Cadmium (Cd 2+ ) interferes with the uptake of water and nutrients and causes injuries to plant roots (Jibril et al., 2017). Wang et al. (2015) reported that Cd 2+ stress decreases total root length (TRL), root surface area (RSA) and root volume (RV) in soybean cultivars. The decrease in root length of lettuce plants due to Cd 2+ stress was as high as 89% (Bautista, Fischer & Cárdenas, 2013). The results of our experiment also showed that the addition of 100 µM CdCl 2 to the nutrient solution decreased the TRL, RSA, RV and root projected area (RPA) of the lettuce genotypes. The numbers of root tips (NRT) and root forks (NRF) in the studied lettuce genotypes were also decreased. This resulted in a decrease in the size of the root systems in all four genotypes. The negative correlations observed among some of the root morphological indexes under Cd 2+ stress was indicative of the adverse effect of Cd 2+ on the morphological indexes of lettuce roots. The tolerance of plants to Cd 2+ stress can be influenced by the characteristics of the roots. Wang et al. (2015) reported that soybean cultivars with larger root systems were tolerant to Cd 2+ whiles those with smaller root systems were sensitive. However, the results of our experiment showed that Yidali, which possessed the smallest root system, had the greatest root tolerance to Cd 2+ Notes. ** Correlation is significant at the 0.01 level (2-tailed). *Correlation is significant at the 0.05 level (2-tailed). The correlation analysis were done using the three values (three replications) for every trait × cultivar × treatment. TRL, total root length; RSA, root surface area; NRF, number of root forks; NRT, number of root tips; RPA, root projected area; RV, Root volume; ABA, abscisic acid; GA3, gibberellic acid; Cyto, cytokinin; IAA, indole-3-acitic acid. compared with the other genotypes that had larger root systems. The tolerance of Yidali to Cd 2+ stress which was demonstrated by the greatest tolerance indexes for RV, RSA, RPA, NRF and NRT could largely be attributed to the diminutive nature of its root system. The smaller root system of Yidali (especially fewer root tips and smaller surface area), probably limited the amount of Cd 2+ absorbed by the roots. Liu, Probst & Liao (2005) attributed the lower Cd 2+ contents in the roots of S. alfedii to the smaller size of root system (smaller root length, surface area and volume). Huang et al. (2015) also indicated that pepper cultivar with shorter roots, fewer root tips and smaller surface area had lower capacity for Cd 2+ uptake. Moreover, the tolerance of plants to Cd 2+ stress has also been assessed based on the content of H 2 O 2 produced when the plants were exposed to the stress. Cd 2+ toxicity results in increased production of reactive oxygen species such as H 2 O 2 and superoxide anion which causes oxidative stress (Tripathi et al., 2017). Zhang et al. (2009) et al., 2019). Our experiment also showed that Cd 2+ toxicity increased the H 2 O 2 contents in the roots of the four lettuce genotypes. However, the increase in H 2 O 2 was least in Yidali, and this also gives an indication of the tolerance of Yidali to Cd 2+ stress compared with the other genotypes. These results, therefore, suggest that lettuce genotypes with smaller root systems could be more tolerant to Cd 2+ stress compared with genotypes that possess larger root systems.
Research studies have shown that almost all hormones have the capacity to act as components of Cd 2+ -stress signaling in plants. The content of cytokinin (Gangwar et al., 2014), abscisic acid (Kim et al., 2014), gibberellin (Zhu et al., 2012), brassinosteroid (Villiers et al., 2011 and jasmonic acid (Zaid & Mohammad, 2018;Noriega et al., 2012) in plants have all been found to change in response to various stresses. Cd 2+ stress increases the levels of ABA (Kim et al., 2014;Stroinski et al., 2010;Fediuc, Lips & Erdei, 2005) and GA (Zhu et al., 2012) in plants. However, the levels of IAA (Gangwar et al., 2014) and cytokinin (Veselov et al., 2003) decreased in plants under Cd 2+ stress. In our study, the effect of Cd 2+ on the contents of the hormones in the roots depended on the genotype. Under Cd 2+ stress, ABA increased in Anyan and Lüsu, decreased in Lümeng and remained unaffected in the Yidali genotype. Moreover, under Cd 2+ stress, the contents of cytokinin increased in Anyan and Lüsu but it decreased in Yidali and Lümeng. The variations in the hormonal responses to Cd 2+ among the four genotypes could be attributed to differences in their sensitivity to Cd 2+ stress. Different lettuce cultivars were found to have exhibited different levels of sensitivity to heavy metals (Priac, Badot & Crini, 2017). Hsu & Kao (2003) also found that the contents of ABA increased in the roots and leaves of Cd 2+ -tolerant rice cultivar but not in the Cd 2+ -sensitive cultivar.
Under the control condition, ABA and IAA contents in roots correlated positively with most of the root indexes whiles GA 3 and cytokinin correlated negatively with TRL and RPA respectively. ABA generally acts as growth inhibitor under stress free conditions (Takezawa, Komatsu & Sakata, 2011). In our study, ABA correlated positively with most of the root morphological indexes under Cd 2+ stress. This suggests that ABA can play a positive role in promoting the tolerance of lettuce plants to Cd 2+ stress. This observation could be attributed to the low concentrations of ABA in the roots of the genotypes tested in the current experiment. Barlow & Pilet (1984) reported that low concentrations of ABA increased root elongation, cell division and DNA synthesis whiles high concentration decreased root growth in maize. The promotion of root growth due to auxin was also demonstrated when root morphological parameters increased upon the application of 5-20 ppm IAA to grafted cucumber seedlings (Balliu & Sallaku, 2017). Although both cytokinin (Mok, 1994) and GA 3 (De Souza & MacAdam, 2001) promote growth through cell division, cell expansion and cell elongation in this experiment. These hormones correlated negatively with TRL and RPA, respectively. This suggests that increasing cytokinin and GA 3 contents in the roots of lettuce under Cd 2+ free condition may decrease root growth. However, under Cd 2+ stress, GA 3 andIAA contents of roots correlated negatively with some of the root indexes but ABA correlated negatively with most of the root indexes compared with GA 3 andIAA. Although under the control condition, both GA 3 and IAA correlated positively with root morphological indexes, both hormones correlated negatively with the root indexes under Cd 2+ stress. This suggests that the interaction effect of these hormones may not influence root growth in lettuce but could enhance the tolerance of the plants to Cd 2+ stress. The positive correlation between ABA and GA 3 and also between ABA and IAA under Cd 2+ stress is indicative of the importance of ABA in alleviating Cd 2+ stress in lettuce plants. ABA is reported to play significant role in plants response to various stresses (Kiba et al., 2011). In plants under drought stress, ABA alleviated the stress by inducing stomata closure, leaf folding and osmotic adjustment (Lipiec et al., 2013). Under stress free conditions, Cytokinin generally suppresses growth and development of roots (Werner et al., 2003) and it is usually antagonistic with IAA in roots (Nordström et al., 2004). However, in this experiment, only cytokinin correlated positively with most of the root indexes under Cd 2+ stress. The positive correlation between cytokinin and most of the root morphological indexes suggests that increased level of cytokinin may be required to support the survival of lettuce plants under Cd 2+ stress. Vitti et al. (2013) found that cytokinin increased root growth of A. thaliana seedlings and improved their tolerance to Cd 2+ stress.
The antioxidant enzymes activities and proline response of plants to heavy metal stress depends on species, cultivar, Cd 2+ concentration and duration of exposure. The enzymatic defense system in plants includes SOD, CAT, and ascorbate peroxidase (Foroozesh et al., 2012). The SOD is a major scavenger which catalyzes the conversion of superoxide to H 2 O 2 and molecular oxygen under stress conditions (Allen, 1995). The H 2 O 2 which is also toxic to plant cells is detoxified by CAT and/or POD, which converts it to water and oxygen (Zhu et al., 2004). Proline is also among the components of the non-specific defense systems in plants. It alleviates metal toxicity by acting as a metal chelator and also as a protein stabilizer (Sharma & Dubey, 2005). In the current experiment, antioxidant enzymes activities and proline response to Cd 2+ stress in the roots of the four lettuce genotypes were similar. Although the SOD activity of the genotypes decreased at seven days after exposure to Cd 2+ stress, the CAT and POD activities, as well as the contents of proline increased. The decrease in SOD activity observed in all the genotypes at seven days after the Cd 2+ treatment could be due to fact that the SOD which is the first enzyme involved in the antioxidant enzyme defense system might have completed its function within few days after the Cd 2+ treatment, which resulted in the increased H 2 O 2 contents. The increase in CAT and POD activities was to enable the plants further detoxify the H 2 O 2 which is also toxic to plant cells. Decreased SOD activity due to Cd 2+ stress was reported in lettuce (Xu et al., 2014), A. thaliana (Abozeid et al., 2017) and in P. sativum L. (Rodriguez-Serrano et al., 2006). The CAT and POD activities and the contents of proline in the roots of the lettuce genotypes exposed to Cd 2+ increased to counteract the Cd 2+ -induced oxidative stress (increased H 2 O 2 contents) in the plants. In a previous experiment, POD, SOD and CAT activities and the content of proline in oilseed rape plants exposed to Cd 2+ stress increased (Yan et al., 2015). In other experiments, the contents of proline in different lettuce varieties increased (Jibril et al., 2017) and the POD activity in pea seedlings also increased (Agrawal & Mishra, 2007) increased when these plants were exposed to Cd 2+ stress.

CONCLUSIONS
In this study, the root tolerance and biochemical response of four lettuce genotypes to100 µM CdCl 2 (Cd 2+ ) treatment were determined. The results showed that Cd 2+ stress decreased the sizes of the root systems of the four lettuce genotypes evaluated. However, the Yidali genotype, which possessed the smallest root system, exhibited the greatest tolerance to Cd 2+ stress with the by exhibiting the greatest tolerance index scores for root volume, surface area, projected area and numbers of root forks and root tips. Cd 2+ stress also caused increases in H 2 O 2 contents in the roots of the genotypes but the increase was least in the Yidali genotype which showed more root tolerance to Cd 2+ stress. The effect of Cd 2+ stress on ABA, IAA, GA 3 and cytokinin was genotype dependent. Under Cd 2+ stress, the correlation between ABA and IAA, GA 3 and cytokinin were positive. The antioxidant enzyme activities and proline response to Cd 2+ stress in the roots of the four genotypes were similar. The SOD activity decreased whiles the CAT and POD activities, as well as the contents of proline, increased under the stress condition. Greater root tolerance indexes and lower H 2 O 2 content were found in the Yidali genotype under the stress condition, suggesting that lettuce genotypes with smaller root system could be more tolerant to Cd 2+ stress. However, further research is needed to ascertain the details of the molecular and genetic mechanisms of Cd 2+ tolerance in lettuce genotypes with relatively smaller root systems.

ADDITIONAL INFORMATION AND DECLARATIONS Funding
This research was funded by the National Natural Science Foundation of China (No. 31660584 and No. 31260473), the China Agriculture Research System (CARS-23-C-07) and the Major Science and Technology Projects Fund of Gansu Province (No. 17ZD2NA015). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.