Physiological And Molecular Mechanisms Of Chromium Stress And Tolerance In Rice

Chromium occurs naturally in several oxidation states, with the trivalent (Cr3+) and hexavalent (Cr6+) forms being the most stable and common. Although Cr3+ is considered as an essential element for human and animals, both Cr3+ and Cr6+ are toxic to plants. Hexavalent chromium is known to be much more toxic than trivalent chromium to living organisms, and is easily reduced to Cr3+ by redox reaction. In comparison with some other toxic metals, like Cd and Pb, Cr has received relatively little attention from plant scientists. Its complex electronic chemistry and changeable station have been a major factor affecting the research on its toxicity mechanisms in plants. Chromium phyto-toxicity has been intensively studied from the multiple levels, including plant growth and development, physiological processes, antioxidant system. However, there is little information on the mechanisms and genotypic difference of Cr toxicity and tolerance in plants. The current experiments were carried out to investigate the physiological and molecular mechanisms of Cr toxicity and tolerance in rice plants, based on the research on chromium availability in soil, Cr toxicity in rice plants, Cr kinetic uptake, subcellular distribution and chemical forms of Cr in plant tissues, and the proteomic changes in responses to chromium toxicity. Meanwhile, the possibility of reducing Cr uptake and alleviating Cr stress in rice plants by exogenous application of chemical regulators were also studied. The major results are summarized as follows:1. The influence of pH and organic matter content in paddy soil on heavy metal availability and their uptake by rice plantsSoil pH, organic matter content and EDTA-extractable heavy metal contents in 27 paddy soils from three locations of Zhejiang province and the heavy metal concentrations in rice plants were investigated to elevate the influence of soil properties on heavy metal uptake and translocation in rice plants. The results showed that the soils from Nanhu exhibited the highest soil organic matter content and EDTA-extractable heavy metal contents, but the lowest pH value. Simple linear regression analysis suggested that the EDTA-extractable contents of Cr, Cu, Fe, Mn, Pb and Zn was negatively correlated with soil pH value, but positively correlated with organic matter content. The combination of soil pH and Log10(OM) in the stepwise multiple linear regression analysis could produce a more precise model for estimation of EDTA-extractable Cu, Pb and Zn contents in soils, but not for Cr, Fe and Mn, indicating that availability of heavy metal in soil was a complex parameter, and determined by many soil factors. Rice plants grown in Nanhu soil had the higher straw heavy metal concentrations than those grown, coinciding with the results of soil EDTA-extractable heavy metal content. Simple linear regression analysis suggested that heavy metal concentrations in rice straw and grains were negatively correlated with soil pH value, but positively correlated with soil organic matter content, except grain Pb and Zn concentrations. Stepwise multiple linear regression analysis indicated that soil pH played an important role in predicting heavy metal concentrations in rice plants, suggesting that it is possible to reduce the heavy metal accumulation in rice plants by regulating the pH level in paddy soil through agronomic approaches.2. Genotypic and environmental variation in chromium, cadmium and lead concentrations in rice grainsGenotypic and environmental variation in Cr, Cd and Pb concentrations of rice grains and the interaction between these metals were investigated by using 138 rice genotypes grown in three contaminated soils. There are significantly genotypic differences in the three heavy metal concentrations of rice grains, with the absolute difference among 138 rice genotypes in grain Cr, Cd and Pb concentrations being 24.5,9.1 and 23.8 folds under the slightly contaminated soil (Cr, Cd and Pb content was 4.61,1.09 and 28.28 mg kg-1, respectively), respectively. Some genotypes, such as Xiushui 113, Zhongjian 9836 and Yongdan 24 etc. which showed consistently low grain Cr, Cd or Pb concentration under the 3 contaminated soils were identified. There was a highly significant interaction between genotype and environment in the 3 heavy metal concentration of the rice grains, suggesting the importance of cultivar choice for a given environment. Correlation analysis showed that Cr concentration in rice grains was not correlated with Cd and Pb concentration in the three contaminated soils. However, there was a significant correlation between Cd and Pb in slightly and highly contaminated levels. The results indicated that interaction between heavy metals in their availability in soil and accumulation in plant is complex.3. The influence of chromium toxicity on antioxidant system and nutrient uptake and accumulation in rice plantsThe effect of chromium (Cr) on the lipid peroxidation, activities of antioxidant enzymes, and the uptake and accumulation of nutrient were studied in two rice genotypes, Xiushui 113 and Dan K5, differing in grain Cr accumulation. The treatment with low Cr level (10μM) showed little influence on lipid peroxidation but increased the activities of SOD and POD. However, Cr stress with high level (100μM) significantly increased MDA content and decreased the activities of SOD and POD in both leaf and root, suggesting that high chromium level in medium would induce serious oxidative to rice plant. Furthermore, chromium toxicity significantly decreased the uptake and distribution of nutrients in rice plants. Maximum nutrient accumulation occurred at level of 10μM Cr, while the minimum accumulation occurred at level of 100μM Cr, indicating more plant growth at 10μM Cr than the control and other Cr treatments. It may be assumed that there might be a synergistic effect of Cr on the plant growth in micro doses. Chromium accumulation was significantly and negatively correlated with the accumulation of each nutrient, suggesting that increasing Cr level may create nutrient deficiencies or imbalance in rice.4. Changes of organic acid exudation and rhizosphere pH in the rice plants under chromium stressThe changes of rhizosphere pH, organic acid exudation of roots under chromium stress and their effects on chromium uptake and accumulation were studied using two rice genotypes though determining organic acid contents and checking the solution pH values. The results showed that rhizosphere pH increased with Cr level in the culture solution and the exposed time. The effects of Cr level and exposed time on organic acid exudation varied with the species of organic acids as well as genotypes. Among the 6 organic acids examined in this experiment, oxalic and malic acid contents were much higher than citric, latic, acetic and succinic acid, and had significantly positive correlation with rhizosphere pH, indicating that the exudation of these organic acids might play an important role in the change of rhizosphere pH. In addition, Cr accumulation in rice plants showed significantly positive correlations with rhizosphere pH, oxalic, malic and citric acid contents, respectively. It may be suggested that increase in rhizosphere pH, and oxalic, malic and citric acid exudation enhances Cr accumulation in rice plants.5. Kinetic characteristics of chromium uptake by rice rootThe kinetic characteristics of Cr uptake by rice roots for both Cr3+ and Cr6+ were studied with hydroponic culture and different Cr supply concentrations and exposure time. The results showed that the uptake rate of both Cr3+ and Cr6+ by rice root increased with the Cr supplying level, but they were saturated at high Cr level (Cr3+,100μM; Cr6+ 400μM). When fitted to Michaelis-Menten equation (V= Vmax* C/(Km+C)), the kinetic uptake of Cr3+ and Cr6+ for two rice genotypes could be illustrated as the following equations:for Xiuhui 113, V(Cr3+)= 2295.99* C / (34.78+C), R2= 0.9785**; V(Cr6+)= 853.24* C/(273.36+C), R2= 0.9701**; for Dan K5, V(Cr3+)= 2777.83* C (38.60+C), R2= 0.9438**; V(Cr6+)= 1232.00* C/(550.09+C), R2= 0.9915**. Hexavalent chromium showed much higher Km value but much lower Vmax than Cr3+, suggesting that Cr3+ had much more affinity with rice roots than Cr6+. In addition, both Km and Vmax values of Dan K5 were much higher than those of Xiushi 113, indicating that Dan K5 had lower affinity with Cr but higher potentiality to uptake Cr than Xiushui 113. During 0.5-48h with supplying 100μM Cr3+ and Cr6+, Cr concentration in rice root increased with the exposure time. However, Cr3+ concentration was saturated at t=12h, while Cr6+ was not saturated at that time. Furthermore, the uptake of Cr3+ was more rapid than of that of Cr6+. For an example, at 0.5h after treatment, Cr concentration in rice roots rose up to 400μg g-1 DW when supplied with Cr3+, while only 20μg g-1 DW when supplied with Cr6+. The experiment of metabolic inhibitors and low temperature showed that the uptake Cr6+ was energy dependent, but Cr3+ required much less energy. It may be assumed that that Cr6+ uptake is an active process, while Cr3+ uptake is passive one.6. Subcellular distribution and chemical forms of chromium in rice plants under different chromium stressThe subcellular distribution and chemical forms of different heavy metals in rice is correlated with their bio-toxicity. An experiment was conducted to investigate the subcellular distribution and chemical forms of chromium (Cr) in two rice genotypes (Oryza sativa L. cvs. Xiushui 113 and Dan K5) differing in Cr accumulation, to understand the mechanism of Cr toxicity and tolerance in rice plants. The results of microbeam synchrotron radiation X-ray fluorescence (μ-SRXRF) showed that Cr in rice tissues mainly distributed in epidermis and vascular bundles, indicating that the epidermis and xylem play important roles in Cr fixation and translocation, respectively. Furthermore, it was found that Cr in the root cells of rice plants exposed to Cr stress was mainly localized in cell walls, whereas Cr in leaf or stem cells were mainly present in both cell walls and vacuoles, suggesting that both compartments act as important protective barriers against Cr toxicity in rice cells. Although Cr ions in all plant tissues exist predominantly in the forms extracted by 80% ethanol and d-H2O, the amount of Cr in the chemical forms extracted by 2% HAc,0.6M HCl and in residues was significantly increased under the highest Cr level (100μM Cr) compared to the plants grown under lower Cr levels. These results also indicate that excess Cr accumulated in rice plants under Cr stress is bound to undissolved or low bioavailable compounds, such as undissolved phosphate and oxalate, being beneficial for rice plants to alleviate Cr toxicity. In addition, under the highest Cr level (100μM), Dan K5 had a higher percentage of Cr in the chemical forms extracted by 2% HAc,0.6μM HCl and in residues compared to Xiushui 113 in both stems and leaves, indicating that more Cr ions in shoots of Dan K5 were bound to undissolved or low bio-available compounds, in comparison with those of Xiushui 113. It is evident that the low bioavailability of Cr in the shoots of Dan K5 is related to a high Cr accumulation.7. Proteomic changes in response to chromium treatments in riceThe mechanisms of chromium toxicity and tolerance still remain to be illustrated. In the present study, an investigation aimed at understanding molecular mechanisms of chromium toxicity was carried out using proteomic analysis. The seedlings of two rice genotypes (Oryza sativa L. cvs. Xiushui 113 and Dan K5) differing in Cr accumulation were exposed to three Cr treatments from 0 to 200μM. Proteins were extracted from the leaves and roots collected from both control and stressed seedlings. Two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry was adopted to investigate the protein expression patterns of rice leaves and roots in responses to Cr stress. A total of 55 proteins (41 in leaves and 14 in roots) were identified in response to chromium stress by MALDI-TOF mass spectrometry analysis, containing RuBiCo, proteins involved in energy and material metabolism, and the potential proteins involved in Cr tolerance. A group of novel proteins were identified to up-regulate in responding to Cr stress, including Hsp90, NADP-isocitrate dehydrogenase, S-adenosylmethionine synthetase, glyoxalase I, reversibly glycosylated polypeptide, Glutamine synthetase, ATP synthase, Guanine nucleotide-binding protein beta subunit-like protein and Signal recognition particle 54 kDa. These proteins are involved in several cellular processes, including cell wall synthesis, energy production and metabolism, electron transport and detoxification. A protein, named reversibly glycosylated polypeptide, which is involved in cell wall synthesis, was the first time being found associated with heavy metal stress.8. Effect of glutothione in alleviating chromium toxicity to rice plantsThe effect of exogenous glutathione (GSH) in alleviating chromium stress was estimated through examining plant growth, chlorophyll and soluble protein contents, antioxidant enzyme activity and lipid peroxidation in rice seedlings exposed to chromium stress. The results showed that plant growth, and chlorophyll and soluble protein contents were severely reduced when the rice plants were exposed to 100μM Cr. Addition of GSH in the culture solution dramatically alleviated the reduction of plant growth, and chlorophyll and soluble protein contents. The activities of some antioxidant enzymes, including SOD, CAT and GR in rice leaves and CAT and GPX in rice roots were increased under Cr stress, which is attributed to defending response to oxidative stress in plants. Addition of GSH reduced the MDA accumulation and increased the activities of antioxidant enzymes in both leaves and roots, suggesting that GSH may enhance antioxidant capacity in Cr-stressed plants. Furthermore, GSH addition significantly decreased Cr uptake and root-to-shoot transport in rice plants exposed to Cr stress. It can be assumed that GSH is involved in Cr compartmentalization in root cells.9. Effect of silicon in alleviating chromium toxicity to rice plantsThe alleviatory effect of Si on Cr toxicity to rice was investigated using a hydroponic experiment with 12 factorial treatments of two Cr levels (0 and 100μM), three Si levels (0,75 and 150mg L-1) and two rice genotypes (Dan K5 and Xiushui113). The results showed that 100μM Cr markedly decreased plant height, dry biomass, soluble protein content, and root antioxidant enzyme activity, whereas significantly increased Cr concentration and MDA content. However, the reduction of plant height, dry biomass and soluble content was greatly alleviated from Si addition to the hydroponic solution. Compared with the plants treated with Cr alone, Si addition significantly reduced Cr uptake and translocation in rice plants. No significant difference was observed between the two Si treatments (75 or 150mg L-1) in shoot Cr concentration and Cr translocation factor. Si addition also alleviated the reduction of anti-oxidative enzymes SOD and APX in leaves; CAT and APX in roots and the increase of MDA content in the Cr-stressed plants. Furthermore, the beneficial effects of Si on activities of anti-oxidative enzymes under Cr stress were genotype dependent. The highest activities of SOD, POD, CAT and APX in leaves occurred in the treatment Cr+Si 150 for Xiushui113 and in the treatment Cr+Si 75 for Dan K5, respectively. It may be suggested that the beneficial effect of Si on alleviating oxidative stress was much more pronounced in Dan K5 than in Xiushui113. The current results showed that Si alleviates Cr toxicity through lowering Cr uptake and translocation and enhancing the activities of anti-oxidative enzymes.