Foliar application of abscisic acid mitigates cadmium stress and increases food safety of cadmium-sensitive lettuce (Lactuca sativa L.) genotype

Plant materials and growth conditions

The study was conducted at the College of Horticulture, Gansu Agricultural University, Lanzhou, Gansu Province, PR China (36°03′N, 103°40′E). The seeds of Cd²⁺-sensitive lettuce cultivar (Lactuca sativa L., cv. Lüsu), purchased from Gansu Academy of Agricultural Sciences, Lanzhou, China, were used in this experiment. The Lüsu genotype was identified as a Cd²⁺-sensitive genotype among other genotypes in our previous studies (Dawuda et al., 2019). The seeds were germinated in a triple-layer filter papers lined in petri dishes within 48 h under light condition in a climate box (temperature 20 °C; light intensity 200 µmol m⁻² s⁻¹ photosynthetic active radiations, PAR; relative humidity 80%). The germinated seeds were sown in trays filled with commercially produced substrate and the seedlings were nursed under greenhouse conditions (average daily temperature 24 ± 2 °C; relative humidity 60–70%; 12 h light) for 14 days. During the first week of growth, the young seedlings were irrigated with 1/4-strength Hoagland’s nutrient solution (Hoagland & Arnon, 1950) with pH 5.5. After the first week, watering was done using $\frac{1}{2}$ strength of the Hoagland’s nutrient solution until the seedlings were 28 days old.

The 28 days old seedlings were transferred to Hoagland’s nutrient solution and allowed for four days to acclimate 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) before treatment application. In all, 288 seedlings were grown in 72 opaque hydroponic containers (1.5 L capacity each). Each of the containers was filled with 1.2 L of full-strength Hoagland’s nutrient solution and supplied with 4 seedlings. The nutrient solution was changed at 4 days interval until the plants were harvested for data collection.

Experimental design and treatments

The experiment was conducted in a climate box using 28 days old seedlings. Five treatments (Table 1), including (i) Control, (ii) 100 µM Cd (CdCl₂), (iii) 10 µg L⁻¹ ABA, (iv) 0.01 g L⁻¹ ABA-inhibitor (fluridone) and (v) CdCl₂+ABA were applied in a completely randomized design with 3 replications. We used the 100 µM CdCl₂ based on our previous experiment in which we evaluated the response of four lettuce genotypes to Cd²⁺ stress (Dawuda et al., 2019). The concentration of ABA used in this experiment was chosen based on previous experiment (Al-Muhairi et al., 2015; Al-Muhairi et al., 2016). The concentration of fluridone (C₁₉H₁₄F₃NO, 99.4%, Sigma-Aldrich, USA), which is ABA-inhibitor, was also chosen based on previous studies conducted by Vysotskaya and co-authors (Vysotskaya et al., 2018). The 100 µM CdCl₂ treatment was supplied through the hydroponic nutrient solution while the ABA and fluridone treatments were sprayed on either side of the leaves. Each group of four plants per pot was sprayed uniformly with 10 ml (approximately 2.5 ml per plant) of either ABA or fluridone depending on the treatment. The CdCl₂ treated plants and the control plants were sprayed with distilled water in a similar manner as the ABA and fluridone treatment. The plants were sprayed for four days at 24 h intervals and then monitored for a further 10 days before the experiment was ended.

Each experimental unit consisted of three plastic containers (1.5 L capacity each). Each container was supplied with 4 plants and a total of 36 plants were used per treatment. The control plants were grown in normal Hoagland’s nutrient solution. The ABA, fluridone and distilled water were applied using separate 100 mL capacity hand sprayer. The treatments were applied to the 28 days old seedlings. The first spraying of plants with ABA, fluridone or distilled water was done just before the plants were exposed to 100 µM CdCl₂ treatment for the first time. Leaf and root samples were collected at 10 days after start of treatments for the determination of lipid peroxidation, antioxidant enzymes activities and photosynthesis indexes. Data on the contents of nutrient elements and cadmium, as well as the biomass of the plants were measured at 14 days after treatments.

Data measurements

Root and leaf samples were collected from four plants which were randomly selected per replicate at 10 days after the start of treatment application for the determination of biochemical parameters including lipid peroxidation and antioxidant enzymes activities. The contents of photosynthetic pigments, gas exchange parameters and relative water content of leaves were also measured at 10 days after the start of treatments. The contents of nutrient elements and Cd²⁺, as well as the root and leaf biomass of the plants were measured at the end of the experiment (14 days after start of treatments) using six plants per replicate.

Determination of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) contents

Contents of H₂O₂ in the roots and leaves samples were determined following the procedure described by Junglee et al. (2014) with slight modifications. Briefly, 0.1 g fresh samples were ground with liquid nitrogen in mortar with pestle and the homogenate was transferred into two mL capacity centrifuge tubes which were then kept in ice bath. We added 1.5 mL 0.1% trichloroacetic acid (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 one 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 H₂O₂ were calculated using the H₂O₂ reference standard curve (0, 1, 2, 3, 4 and five mmol L⁻¹).

Contents of MDA in leaf and root samples were measured as described by Fazeli, Ghorbanli & Niknam (2007) with some modifications. In brief, the fresh leaf and root tissues (0.3 g each) were ground separately in ice bath with two mL 0.05 mol L⁻¹ TCA. The homogenate was centrifuged at 10,000 g for 5 min at 25 °C and then we added five mL 0.5% (m/v) thiobarbituric acid (TBA) solution to the supernatant. The extract was placed in a hot water bath for 10 min, and then quickly moved to cold water bath. After cooling, the solution was centrifuged again at 10,000 g for 10 min at 25 °C. The absorbance of the supernatant was measured at 532 nm; and the value for nonspecific absorption at 600 nm was subtracted. The content of MDA was calculated from the molar extinction coefficient of MDA-TBA at 155 mmol⁻¹ cm⁻¹.

Determination of antioxidant enzymes activities

Root and leaf samples were collected at seven days after treatments application and stored at −80 °C for enzymes assays. 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 aliquot for 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 supernatants were used for the determination of enzymatic activity. The SOD, POD and CAT activities were measured as described by Giannopolitis & Ries (1977), Chance & Maehly (1955) and Nakano & Asada (1981), respectively.

Determination of contents of chlorophyll and carotenoids

In brief, leaf tissues were collected from six plants per treatment at 10 days after the start of treatments and 0.1 g (fresh weight) was ground in 10 ml 85% acetone. The absorbance or optical density (OD) values for the extracts were measured at 663, 645 and 440 nm after 48 h using a spectrophotometer (UV 1800, Tokyo, Japan) and the contents of chlorophyll a, chlorophyll b, chlorophyll a+b and carotenoids were measured following the method of Zhang (1992).

Determination of gas exchange parameters

Net photosynthetic rate (P_N), stomatal conductance (Gs), transpiration rate (Tr) and intercellular concentration of CO₂(Ci) were determined using a portable photosynthetic system (CIRAS-2, PP System, UK) following the method of Gu et al. (2007) with slight modification. In brief, two fully expanded leaves from the top of two plants per treatment were selected for the measurement. The photosynthetic photon flux density (20000 lux), ambient CO₂concentration (380 µmol mol⁻¹), leaf temperature (22 °C) and relative humidity (65–75%) were maintained in the climate box for period of the measurements.

Determination of relative water content

The relative water content (RWC) was measured as described by Hayat et al. (2007). In brief, fully expanded leaves were collected from each treatment and four fresh leaf discs (two cm diameter, excluding midrib) were taken, weighed and immediately floated on deionized water in petri dishes. Each petri dish was supplied with 100 ml deionized water to saturate the leaf disc for 24 h, under dark condition. At 24 h, the leaf discs were removed from the water and blotted with tissue papers to remove the adhering water and the turgor mass recorded. The leaf discs were then dried at 70° C for 48 h. The RWC was calculated as follows: ${\displaystyle \mathrm{RWC}=\frac{\mathrm{FW}-\mathrm{DW}}{\mathrm{TW}-\mathrm{DW}}\mathrm{X}100}$ where, FW is fresh leaf weigh; DW is dry leaf weight; TW is turgor weight of leaf.

Determination of contents of nutrient elements and cadmium

The nutrients and Cd²⁺ contents were measured according to the method of Azevedo et al. (2005) with some modifications. In briefly, leaf and root samples were collected from three lettuce plants for each treatment and the fresh weights determined. The samples were washed several times in deionized water, put in envelopes and dried at 80 °C until the dry weight remained constant. The dried samples were ground and sieved with 0.25 mm mesh. The fine samples (1.0 g each) were put into crucibles and burnt to ash at 550 °C for 5–6 h. The ash samples were allowed to cool and one mL nitric acid solution was added and mixed thoroughly. The mixture was then filtered and the volume made up to 50 mL by adding drops of distilled water. The concentrations of the elements were determined using the atomic absorption spectrophotometer with the OD set as follows: 422.6 nm (Ca); 285.2 nm (Mg); 324.8 nm (Cu); 248.3 nm (Fe); 213.8 nm (Zn); 228.8 nm (Cd). The contents of the nutrient elements (leaf) and cadmium (leaf and root) were calculated using the following formula:

ω = ((c-c₀) ×50 ×N ×1000) / (m ×D)

where:

ω: Content of nutrient element

c: Concentration of elements in sample solution (mg/L)

c₀: Concentration of elements in blank control solution (mg/L)

N: Dilution multiple

m: Weight of sample (g)

D: Is a constant, when the value is represented by mg/kg, D is 10³; when the value is represented by g/kg, D is 10⁶.

50: The volume of the sample solution.

Plant biomass and biomass response to stress (BRS)

The fresh leaf and root biomass per plant were measured immediately after harvest at 14 days after the start of the treatments. The leaf and root samples were put in separate envelops and dried at 65 °C for 48 h for the dry weights. The biomass response to stress (BRS) which gives an indication of the tolerance of the plant or plant organ to stress was calculated as described by Wilkins (1978) with slight modification. The BRS was calculated using the average plant weight of each treatment at 14 days after start of treatments as follows: ${\displaystyle \mathrm{BRS}=\frac{\text{Plant in stress}}{\text{Plant without stress}}x100.}$

Data analyses

The data measured were subjected to a one-way analysis of variance using Genstat statistical software (12th edition, Lawes Agricultural Trust, Rothamsted Experimental Station, UK). The treatment means were separated by the least significance difference (LSD) test at 5%. The results are presented as means + standard deviations. Different letters assigned to the bars indicate significant differences among the treatments by LSD (P < 0.05). Graphs were prepared using Graphpad Prism version 8.0.

Hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) contents

The effect of CdCl₂ on oxidative stress was determined by measuring the contents of H₂O₂ and MDA in the root and leaf tissues of the plants. The H₂O₂ and MDA contents in both roots and leaves were significantly (P < 0.001) affected (Fig. 1A). Relative to control plants, the CdCl₂ treatment increased the contents of H₂O₂ in the roots and leaves by 65.3% and 65.1%, respectively. Moreover, CdCl₂ treatment increased the contents of MDA in the roots and leaves by 48.4% and 44%, respectively (Fig. 1B). However, application of ABA to the plants under CdCl₂ treatment (CdCl₂+ABA) suppressed the accumulation of H₂O₂ in both roots and leaves by about 3.4 and 3.1 folds, respectively. In addition, exogenous ABA inhibited the accumulation of MDA in the roots and leaves of the CdCl₂ treated plants, decreasing it by 2 and 2.5 fold, respectively. The application of ABA to unstressed lettuce plants slightly increased the H₂O₂ contents in the roots (10.5%) and leaves (5.6%). However, the MDA contents in the roots and leaves of the sole ABA treated plants were similar to the control plants. Moreover, the fluridone treatment also increased the contents of H₂O₂ in the roots by 50% and in leaves by 51.6%. Similarly, the contents of MDA increased in the roots and leaves of the fluridone treated plants by 15.8% and 22%, respectively.

Antioxidant enzymes activities

The role of exogenous ABA in the antioxidant enzymes protection of the lettuce plants was determined by measuring the SOD, CAT and POD activities in the leaves and roots of the plants (Fig. 2). Relative to the control plants, the CdCl₂ treatment significantly (P < 0.001) decreased SOD activities in both leaves and roots by 6.7% and 23.7%, respectively. However, the application of ABA to the plants under CdCl₂ treatment resulted in marginal increases in the leaf (4.3%) and root (5.9%) SOD activities. The ABA treated plants had leaf and root SOD activities similar to the control plants. The fluridone treatment also decreased the SOD activities of the leaves and roots of the plants by 7.2% and 24.4%, respectively. The results also showed that the CAT and POD activities of the plants under CdCl₂ stress increased (Fig. 2). The CAT activities in the leaf and root of CdCl₂treated plants increased by 57.1% and 66.2% respectively. The leaf and root POD activities of the CdCl₂treated plants also increased by 19.2% and 21.6%, respectively. However, the leaf and root POD activities of CdCl₂+ABA treated plants were 4.6% and 4.3%, respectively. The CAT and POD activities of the sole ABA treated plants were similar to those of the control plants. The results also showed that the CAT and POD activities of the fluridone treated plants were high and statistically similar to those of the CdCl₂ treatment.

Chlorophyll (Chl) and carotenoids contents

The contents of Chl a, Chl b, Chl a+b and carotenoids were significantly (P < 0.001) affected by the treatments (Fig. 3). Relative to the control plants, CdCl₂ stress decreased the contents of chlorophyll a, chlorophyll b and chlorophyll a+b by 40.3%, 41.1% and 40.7%, respectively. Moreover, CdCl₂ treatment decreased the content of carotenoids by 45.8%. However, the application of ABA to the lettuce plants under Cd²⁺ stress maintained the contents of chlorophyll and carotenoids at levels that were similar to the control plants. The decreases in the contents of Chl a, Chl b, Chl a+b and carotenoids in the Cd+ABA treated plants were only 4.2%, 3.7%, 4.4% and 32.1%, respectively. The contents of Chl a, Chl b and Chl a+b in the sole ABA treated plants were statistically similar to those of the control plants. However, the application of ABA to unstressed plants caused a 50.5% decrease in the content of carotenoids, relative to the control plants. The application of fluridone also decreased the contents of Chl a, Chl b, Chl a+b and carotenoids by 66%, 63.8%, 61.6% and 56.7%, respectively.

Gas exchange parameters

To investigate the role of ABA in regulating photosynthesis in lettuce plants under CdCl₂ stress, we measured net photosynthesis (P_N), stomata conductance (Gs), transpiration rate (Tr) and intercellular CO₂ (Ci). Relative to the control plants, CdCl₂ significantly (P < 0.001) decreased the P_N, Gs and Tr by 75.3%, 66.3% and 53.8%, respectively (Fig. 4). However, the Ci was statistically the same among the control, the CdCl₂, CdCl₂+ABA and fluridone treated plants. The adverse effects CdCl₂ on the gas exchange indexes was reduced by the application of ABA. Relative to the control plants, the decrease in P_N, gs and Tr in the CdCl₂+ABA treated plants were 20.1%, 14.5% and 29.3%, and these were lesser than those caused by the sole CdCl₂ treatment. Moreover, the CdCl₂+ABA treated seedlings had 3.8% higher Ci compared with the control plants. The application of ABA to unstressed lettuce plants caused 16.6% and 15% increases in P _N and Ci respectively, over the control plants. However, the Gs and Tr of the CdCl₂+ABA treated plants and the control plants were statistically the same. The application of fluridone also decreased P_N, Gs and Tr by 56.5%, 13.5% and 20.3% respectively. However, the Ci of the fluridone treated plants and the control plants were statistically the same.

Relative water content (RWC) of leaf

The application of the treatments significantly (P < 0.001) affected the relative water content (RWC) of the lettuce plants (Fig. 5). The control seedlings, the ABA and the CdCl₂+ABA treated seedlings had greater leaf RWCs of 86.5%, 86.4% and 83.5%, respectively. The CdCl₂ and fluridone treated plants had the least leaf RWCs of 72.4% and 67.8%, respectively.

Macro and micro nutrient elements

The results on macro and micro nutrient elements are presented in Table 2. At 14 days after treatment application, the contents of the nutrient elements (except Cu) were significantly (P < 0.001) affected. Generally, the CdCl₂ and fluridone treatments greatly decreased the contents of the nutrient elements when compared with the control plants. The CdCl₂ treatment decreased the contents of Mg, Ca, Zn, Fe and Mn by 41.3%, 31.6%, 54.5% 29.4% and 68.9%, respectively. In most cases, the application of ABA to the unstressed plants maintained the contents of the nutrients elements to levels similar to the control plants. Although the contents of nutrient elements in the Cd+ABA treated plants were in some cases lower than those of the control plants, they were generally higher than those of the sole CdCl₂ treatment. The application of ABA to unstressed plants maintained the contents of the nutrient elements (except Mn) in the leaves to levels similar to the control plants. With the exception of Cu, fluridone treatment also decreased the contents of nutrient elements to levels lower than the control plants.

Cadmium contents

We analyzed the contents of Cd²⁺ in the root and leaf samples from the various treatments (Fig. 6). In comparison with the control plants, the 100 µM CdCl₂ treated plants had 97.7% and 99.5% more Cd²⁺ in the leaf and root of the plants, respectively. Even though the CdCl₂+ABA treated plants also had higher leaf and root Cd²⁺ contents, the increases were relatively lower compared with the sole CdCl₂ treatment. Moreover, the content of Cd²⁺ in the leaves of the CdCl₂+ABA treated plants (0.11 mg. kg⁻¹ FW) was lower than the 0.2 mg kg⁻¹ recommended for leafy vegetables.

Biomass, biomass response to stress (BRS) and morphology of lettuce plants

Relative to the control plants, the 100 µM CdCl₂ treatment significantly (P < 0.001) decreased fresh leaf weight (FLW), fresh root weight (FRW), dry leaf weight (DLW) and dry root weight (DRW) of the plants by 59.9%, 74.6%, 73% and 66.7%, respectively (Table 3). In addition, the biomass response to Cd stress (BRS) of the CdCl₂ treated plants was the least (36.4%). However, the Cd+ABA treated plants had higher BRS (81.2%) compared to the sole CdCl₂ treated plants. Moreover, the Cd+ABA treated plants suffered lesser reductions in FLW (14.9%), FRW (31.1%), DLW (31.2%) and DRW (33.3%) compared with the sole CdCl₂ treated plants. The root and leaf biomass of plants treated with sole ABA were relatively similar to those of the control plants. However, the application of ABA-inhibitor (fluridone), also decreased the root and leaf biomass of the lettuce plants. Furthermore, the CdCl₂ and fluridone treated plants had the least BRS (36.4% and 58.3%, respectively) indicating the plants suffered greater stress from Cd²⁺ and the ABA-inhibitor (Table 2). In comparison with the CdCl₂ treated plants, the Cd+ABA treated plants had higher BRS (%) value of 81.2%, indicating that ABA improved the tolerance of the plants to the Cd-induced stress. Figure 7 shows the appearance of the plants at 14 days after start of treatments. The control and ABA treated plants appeared similar in terms of size. However, the leaves and roots of the sole CdCl₂ treated plants were greatly reduced in size. In addition, the root system appeared relatively brownish in color. Even though the CdCl₂+ABA treated plants were not as large as the control and the sole ABA treated plants, they were relatively larger than the sole CdCl₂ treated plants. Although fluridone (ABA-inhibitor) treatment did not decrease the leaf and root sizes of the plants as much as the CdCl₂, the roots were relatively brownish in color and portions of the leaves appeared chlorotic and bleached.