Rice paddies are contaminated with Cd because of application of phosphate fertilizer and irrigation using mine lechate respectively (1). Rock phosphate contains Cd admixtures, and repeated application of rock phosphate derived phosphate fertilizer caused Cd pollution of agricultural soils (1). Recently, mining related Cd contamination of rice was reported from China and Thailand (2). Volcano derived paddy soil also contained Cd, and resulted contamination of rice with Cd (3). Daily Cd uptake through staple food such as rice caused bone mineralization related Itai-Itai disease in humans (4). So prevention of Cd contamination is a serious environmental concern. Both plant and field-based strategies reported to minimize Cd accumulation in crop plants (1). Selections of low Cd-accumulating cultivars, plant breeding with low Cd-accumulating traits, and transgenic strategies with Cd excluding genes proposed to decrease Cd load in rice. But field applicability of these strategies is questionable. Mineral nutrition of crop plants found to influence Cd accumulation in plants (5). Among nutrients, Ca, Fe and Zn played important role in alleviation of Cd stress in plants (6, 7, 8). Photosynthesis controls biomass productivity of plants (9, 10). Therefore, monitoring consequences of mineral nutrient supplements on photosynthesis under Cd is an important aspect.
Light harvest during photosynthesis accomplished with the help of photosynthetic pigments and metalloproteins (11, 12). Treatment of Cd hindered biosynthesis of chlorophylls, and this effect was one of the main reasons for retardation of photosynthesis under Cd stress (6). Carotenoids are essential for dissipation of excess light energy (12). Low level of Cd exposure increased carotenoids content in plants whereas a higher level of cellular Cd decreased these pigments (13).The blockage of linear electron transport during photosynthesis ended up in the formation of reactive oxygen species and lipid peroxidation in plants (14).Therefore, it is clear that monitoring of photosynthetic light reactions help to understand effectiveness Ca, Fe and Zn treatments on Cd tolerance.
Genetic loci control Cd tolerance in rice plants (15, 16). Distribution of the genetic loci of Cd tolerance showed variation in rice germplasm (3). Transition metal uptake and transport in plants also reported to take place under control of dominant traits (15). Because of above reasons, two rice varieties were chosen in the present study to understand extent of Cd accumulation and Cd stress tolerance during nutrient supplement. The study conducted on sand culture to maintain natural nutrient mobilization capacity of rice varieties. Thus, the approach followed in the present study helps to formulate fertilization methods that can be exploited for developing field level strategies to minimize Cd content in rice.
Materials and Methods
Rice varieties namely, MO16 and MTU 7029 obtained from District Seed Development Centre, Thrissur, Kerala, India, and seed research center, Prof. Jaya Shankar Telangana State Agriculture University, Telangana, India, respectively. MO 16 is a dwarf variety having medium tillering, post-harvest seed dormancy, non-lodging, and resistant to brown planthopper and GM Biotype-5.This variety cultivated in Kuttanad, a below sea level area in Kerala where intermittent flooding and salinity stress occurs. MTU 7029 is a semi-dwarf variety having profuse tillering and resistance to blast disease. This variety cultivated in Godavari western delta of Andhra Pradesh and Gangetic alluvial soils of West Bengal.
Sand for the experiment collected from Meenachil river (9°41’52.46”N, 76°45’51.09”E) and passed through 2.0 mm mesh. The sand washed with fresh tap water until the water becomes transparent, and after that washed with 1.0 N HCl to remove the nutrients held in the sand. This step followed with the washing of sand in deionized water. The pH of the sand (5.8) measured from 20.0 ml water extract of 10.0 g sand using a pH meter (Digisun, Hyderabad). Seedlings raised in germination boxes contain wet filter paper, and seven-day old seedlings chosen for the experiments. Sand (50.0 g) mixed with 20.0 ml of 100.0 % Hoagland media for the growth of seedlings, and the culture maintained for a month.
The dosage of Cd treatment fixed after a series of trials until there existed a morphological difference between control and Cd-exposed plants, particularly, about change in leaf color and plant height. For Cd treatment, 20.0 ml Hoagland solution contains 2.0 mM CdCl2 mixed with 50.0 g sand. The nutrient challenge performed with addition of 2.0 mM Fe-EDTA, 2.0 mM CaCl2, and 2.0 mM ZnCl2 respectively. The content of EDTA maintained equal among all the experimental plants.
Real-time monitoring of chlorophyll fluorescence is a nondestructive method which helps to study the photochemical reactions in leaves. Time-resolved slow kinetics of chlorophyll fluorescence monitored in the present study. For the fluorescence analysis, plants kept 30.0 min in the dark to ensure open state of the reaction centers (6). The leaves exposed to saturation pulse of 6000. 0 μmol photons m−2s−1 for 0.8s to record maximum fluorescence (PAM2500, Heinz Walz, Germany). Light intensity 303 ± 3 μmol photons m−2s−1 used to measure steady-state fluorescence. The quantum efficiency of photosystem II (YII), linear electron transport rate (ETR) and nonphotochemical quenching (NPQ) plotted against time to reveal the influence of various treatments to recover photosynthetic efficiency under Cd stress.
Metal tolerant plants maintained biomass productivity under metal stress. Hence biomass is an important parameter to be considered for analyzing metal toxicity. Plants cleaned with double distilled water and kept in an air dry oven for 7.0 days before measurement of biomass. The dry weight of the individual plants measured with the help of a weight balance (Sartorius, Germany). The biomass expressed as mg dry weight.
Plant pigments measured from acetone – DMSO (1:1) extract of leaf discs. The leaf discs of uniform size made with the help of a paper hole punch and weighed. Leaf discs placed in test tubes containing extraction solvent and kept on orbital shaker at 200.0 rpm in the dark for 12.0 hrs. The optical density of extract recorded at 663.0 nm, 645.0 nm, and 480.0 nm using a UV-Visible spectrophotometer (Shimadzu UV-1800, Japan). The content of pigments (μg/ml) determined using Arnon’s equations for photosynthetic pigments (17). Chlorophyll a = 0.0127 A663 – 0.00269 A645, Chlorophyll b = 0.0029 A663 – 0.00468 A645, Carotenoids = 1000A470 - 3.27[chl a] - 104[chl b])/227.
Malondialdehyde (MDA) produced during lipid peroxidation. Therefore, the extent of lipid peroxidation in leaves determined via estimation of MDA content. Trichloroacetic acid (TCA) (4.0 ml) used to extract MDA from leaf tissues (500.0 mg).The extract mixed with 1.0 ml 2-thiobarbituric acid (0.5 %) and the mixture heated at 95.0 oC for 30 min. The mixture cooled and centrifuge at 10000.0 rpm for 10 min. The supernatant subjected to the measurement of optical density at 532.0 and 600.0 nm (18). Extinction coefficient value of 155.0 mM−1 cm−1 used to calculate the MDA content in the sample, and the result expressed in nmol MDA per gram fresh weight.
Accumulation of Cd in plant determined from the acid digested solutions of plant tissues. The plant parts manually cleaned with 10.0 mM EDTA solution and deionized water respectively. The washed plant kept in a hot air oven at 80.0 oC for drying. Acid digestion of the dried plant material conducted using a mixture of HNO3-HClO4 (3:1) mixture (19). The powder obtained after the digestion of the plant material dissolved in 0.1 M HCl before quantitative estimation of Cd. Amount of Cd in the solution determined with a Poplar leaf NCSDC 73550 standard reference calibrated atomic absorption spectrophotometer (GBC 932, Australia).
Two-way analysis of variance (ANOVA) carried out to distinguish the effectiveness of the nutrient supplements on Cd tolerance in two rice varieties. Duncan’s multiple range test used to explain the statistical significance. Alphabets starting from letter `a` denoted level of statistical significance. The analysis considered significant at P ≤ 0.05.
Cadmium treatment affected biomass productivity (Fig.1, Table 1). Plants subjected to Cd stress had less biomass (57. 0 % in MO16 and 59.0 % in MTU 7029). The supplements of Fe (59.0 %) and Ca (35.0 %) increased leaf biomass under Cd stress. But the root biomass increased only in the presence of Fe (49.0 %). It also noticed that Zn supplement decreased 7.0 % biomass in MTU7029 under Cd stress.
Changes in fresh weight of plants
|Variety||0||2.0||2.0 Fe||2.0 Ca||2.0 Zn|
|MO 16 (mg/g)||18.1+ 0.2a||8.1 + 0.1c||14.2 + 0.2a||12.5 + 0.5b||8.1 + 0.1c|
|MTU 7029 (mg/g)||17.4 + 0.3a||7.2 + 0.4c||14.1 + 0.1a||11.3 + 0.1b||6.7 + 0.3c|
|MO 16 (mg/g)||2.38 + 0.01a||0.91+ 0.01c||1.89 + 0.02b||1.24 + 0.02c||0.91 + 0.03c|
|MTU 7029 (mg/g)||2.12 + 0.02a||0.84 + 0.02c||1.62 + 0.07b||0.91 + 0.04c||0.76 + 0.04c|
Abbreviations: 0.0 and 2.0 represent control and 2.0 mM CdCl2 treatments whereas addition element symbol indicates supplement of respective elements. Alphabets starting from leter a, b, c, d denoted first, second, third and fourth level of statistical significance. The analysis considered significant at P < 0.05, n=6.
Quantum efficiency of PSII
Quantum efficiency of photosystem II indicates the efficiency of pigment-protein complexes to utilize the absorbed photons for the photosynthetic electron transport. The YII value decreased after Cd treatment. Supplements of the nutrients had a progressive effect on the maintenance of YII value during the transition from open state to closed state (Fig. 2a-b). Above effects not seen after 120.0 s among Zn and Ca supplemented plants. But the gain of YII efficiency noticed among Ca supplemented plants after 160.0 s in MO16 variety. The YII value of Fe supplemented plants was superior to control plants between 80.0 and 160.0 s in MTU 7029 (Fig. 2b).
Linear electron transport rate
Linear electron transport (ETR) generates reducing power critical for CO2 fixation. The alleviatory effect of nutrient supplements on ETR lasted up to 120.0 s, and after that the effect persisted only among Fe and Ca supplemented plants (Fig. 2c-d). The long-term alleviatory effect of Ca on ETR observed clearly in MO16 variety. The MTU 7029 variety had a drop in ETR after 100.0 s, and the activity recovered after 160.0s (Fig. 2d).
Excess light energy harvest or the block in electron transport rate generated nonphotochemical quenching (NPQ) in plants. This process is important for the protection of photosynthetic electron transport assembly under metal stress. The NPQ capacity increased in the course of Cd treatment with nutrient supplement (Fig. 2e-f). But there was a decrease in NPQ after 200.0 s among MO 16 plants supplemented with Ca. The longterm NPQ adaptation to Cd stress observed mainly in MTU7029. It is noteworthy that plants grow with Fe supplement had more NPQ capacity.
Chlorophyll and carotenoids are involved photosynthetic light harvest. Chlorophyll a (30.0 %) and Chlorophyll b (49.0 %) decreased after Cd treatment. But the supplement of Fe prevented decrease in chlorophyll due to Cd stress (Fig. 3a-b). Supplement of Ca also increased chlorophyll a (20.0 %) and b (31.0 %) in MO16. But supplement of Zn did not prevent decrease in chlorophylls content under Cd stress (Fig. 3a-d). Chlorophyll a/b ratio increased after Cd treatment (Fig. 3c). Carotenoids content decreased (40.0 %) after Cd treatment (Fig. 3d). But supplement of nutrients resulted in more carotenoid accumulation (69.0 % for Fe, 59.0 % for Ca, 126. 0 % for Zn) under Cd stress.
Malondialdehyde (MDA) is a product formed during peroxidation of lipids. Amount of MDA was higher (106.0 %) in plants after exposure to Cd (Fig. 4). Supplements of Fe and Ca decreased MDA content (45.0 % for Fe, 33.0 % for Ca) under Cd stress. But Zn supplement did not prevented production of MDA in the course of Cd treatment.
Cadmium uptake occurs in plants through transition metal transporters. It found that more Cd accumulate in MTU 7029 plants compared with MO 16. Treatment of Fe decreased Cd accumulation (35.0 %) in the roots (Fig. 5a). Accumulation of Cd in the leaf decreased with both Fe (58.0 %) and Ca (28.0 %) supplements (Fig. 3b). It also noticed that Zn supplement enhance Cd accumulation (up to 6.0 %) in the leaf.
Chlorophyll fluorescence analysis revealed consequences of nutrient supplements on photosynthetic light reactions during Cd stress. Cadmium accumulation retarded photosynthesis in rice plants (19). In the present study, a supplement of Fe and Ca helped to restore biomass under Cd stress. This effect was the outcome of maintenance of photosynthetic performance and lowering of Cd accumulation in leaves respectively. But Zn supplement was not effective in the restoration of biomass under Cd stress.
Chlorophyll a fluorescence decays from a higher to a lower transient steady state level during excitation of a dark-adapted leaf (20, 21). The time taken to reach the steady state level depended on the efficiency of light energy transfer via electron transport chain. The plastoquinone pool completely oxidized in this state, and the reaction center charge separation is maximum. Therefore, the initial phase fluorescence analysis reflects the efficiency of photochemical reaction phase which helps to probe light harvest and electron transport efficacy in the quinines (QA) binding sites (21). The analysis of effective quantum yield of PSII photochemistry (YII) showed a higher value at initial time phase during nutrient supplements during Cd stress. These change indicated that nutrient supplement helped to prevent the adverse effect of Cd on light harvest and electron transfer to plastoquinone. Among the nutrients chosen for the study, Fe supplement had the highest impact on the recovery of initial YII value. This result was the outcome of more chlorophyll a accumulation involved in light harvest (22). The decrease of chlorophyll content account for low initial YII value during Ca and Zn supplements compared with that of Fe. The above results helped to predict that low light harvest due to decrease of chlorophyll content accounts for the very first step in the inhibition of photosynthesis during Cd stress. The low YII value of Cd stressed plants also indicated damage in the electron donor side of PSII under Cd stress (23). Longevity of higher YII indicated the efficiency of photoelectron transfer beyond PSII (21). The time-resolved YII curve indicated that supplement of Fe and Ca helped to uphold YII value for longer periods compared with Zn. The severe decrease of YII after 120.0 s pointed blockage of electron transport at PS I. Above results also indicated that PQ and cytochrome b6f are not be a bottleneck in electron transport under Cd stress (22).
Linear electron transport rate account for the terminal reduction of NADP+ important in CO2 fixation (24). The gradual permanent decrease of electron transport observed in the case of Cd treatment pointed blockage of electron transport at the PSI. Plants subjected to nutrient supplement maintained electron transport rate until 120.0 s, and after that, the rate of electron transport decreased. These results point out blockage of electron transport in the downstream of PSII donor site (25). Secondly, recovery of electron transport observed with Ca supplement before 100.0s indicated Cd-induced damage in Ca binding site of PSII (26).
Nonphotochemical quenching (NPQ) occurs when photon energy not utilized in electron transport and (27). The NPQ capacity of Cd-exposed plants was low because of low carotenoids content that mediate NPQ. The lowering of plant pigments during Cd stress also inhibited both harvesting of the photon and photochemical reactions. The increments in Chlorophyll a/b ratio point out the need of more light harvest capacity under Cd stress. Nutrient supplements increased NPQ, and this change associated with an increase of carotenoids. An increase of NPQ observed with Cd treatments plus Zn supplements confirm the inhibitory action of Cd on electron transfer at PSII (28, 29). It is noteworthy that NPQ decrease after the initial hike because of reoxidation of PQ.
The blockage of electron transport is well known to produce reactive oxygen species in plants (30). The reactive oxygen species react with membrane lipids and result lipid peroxidation in plants (31). Analysis of malondialdehyde content indicated that extent of lipid peroxidation increased with Cd accumulation. Low MDA content in the leaves during Fe and Ca supplements was the result decrease in Cd accumulation. Thus, it concluded that decrease in Cd accumulation accompanied with low level of membrane damage and normal functioning of photoelectron transport account for the alleviation of Cd stress during Fe and Ca supplements.
Cadmium inhibited photosynthetic electron transport, and resulted lipid peroxidation in rice plants. Rice variety MTU 7029 accumulated more Cd compared with MO 16. We have also noticed that photochemical reactions in MTU 7029 inhibited more than MO 16 during Cd stress. Addition of Fe and Ca ions helped to restore photosynthetic performance under applied level of Cd stress. This effect may be the outcome of low Cd accumulation what enable to avoid delterious effects of Cd such as lipid peroxidation of membranes which is known to inhibit photosynthetic electron transport. Secondly, the supplement of Zn did not help to recover photosynthesis light reactions under Cd stress probably because of inability to decrease Cd accumulation. So we concluded that supplementation of Fe and Ca ions to the culture medium help to uphold light reactions of photosynthesis in rice plants during Cd exposure.
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