Mechanisms Of Alleviation Effects Of Glutathione Application On Cadmium Toxicity And Its Genotypic Difference In Barley

Cadmium(Cd)is one of the most deleterious heavy metals to both plants and animals and has no beneficial biological function in the aquatic or terrestrial organism,but can be absorbed and accumulated easily by plants,while high accumulation of Cd in plants not only affects crop yield and quality badly,but also gives rise to a threat on human health via food chain.Although several approaches have been proposed to reduce soil Cd level. including the use of hyper-accumulating plants,none have been effectively applied. Considering large-scale medium/slightly contaminated farmlands such approaches as selection/breeding of crop genotypes/cultivars tolerant to Cd toxicity and with low Cd accumulation in edible parts,the improvement of agronomic practice and application of chemical regulators which can reduce plant Cd uptake would be a cost-effective and practical substitute mode to fully utilize natural resource and guarantee safe food production.Reduced glutathione(GSH)is the direct substrate for PCs synthesis,and the roles of GSH and PCs in heavy metal tolerance were reported by many researchers. Accordingly,the present study was carried out to select Cd- tolerant and sensitive genotypes,and to elucidate the mechanism of mitigative effect of GSH application on Cd toxicity and its genotypic difference by examining growth parameters,photosynthesis, anatomic structure,reactive oxygen species(ROS)metabolism and other physio-chemical responses of the two selected genotypes differing in Cd tolerance.The main results are as follows:1.Two successive hydroponic experments were carried out to identify barley varieties tolerant to Cd toxicity via examing SPAD value,plant height,dry weight,tillers per plant,root length and volume,and biomass accumulation.The results showed that SPAD value,plant height,dry weight,root length and volume,and biomass accumulation were significantly reduced in the plants grown in 20μM Cd compared with control,and Zn,Mn and Cu absorption and translocation were also strictly hindered.There was a highly significant difference in the decline of these growth parameters among genotypes.Weisuobuzhi and Jipi 1 showed the least reduction in both experiments,suggesting their high tolerance to Cd toxicity,while Dong 17 and Suyinmai 2 with the greatest decline and toxicity symptoms appeared rapidly and severely,donoting as Cd-sensitive genotypes.In addition,significant genotype difference in Cd concentration was also found,with Weisuobuzhi containing the highest,and Jipi 1 the lowest Cd concentration in shoots.2.Hydroponic experiments was undertaken,using two selected genotypes Weisuobuzhi (Cd-tolerant)and Dong 17(Cd-sensitive)exposed to 4 treatments of 0μM Cd(control), 5μM Cd(Cd),20 mg L^-1GSH(GSH),and 5μM Cd+20 mg L^-1GSH(Cd+GSH),to study the genotypic difference in the effect of exogenous GSH on barley growth, nutrient uptake and translocation under Cd stress.The results indicated that the tolerant genotype Weisuobuzhi,compared with the sensitive genotype Dong 17,showed much slighter visual leaf Cd toxic symptoms of necrotic patches but with significantly higher Cd concentration in shoots after 25 d 5μM Cd exposure.Cadmium stress seriously inhibited Zn,Mn and Cu absorption and translation in the both genotypes.Addition of 20 mg L^-1GSH(Cd+GSH)significantly reduced Cd uptake and accumulation compared with Cd treatment and being emphasized in Dong 17,but improved Zn,Mn and Cu uptake and its root/shoot concentration was even higher than control.Cadmium toxic symptoms was alleviated even more significantly in Dong 17 by GSH application, and the increases in plant height,root length and dry weight over its Cd treatment were much higher than that in Weisuobuzhi.3.The effect of exogenous GSH on barley cytoarchitecture and photosynthetic characteristics after 15 d Cd treatment and its genotypic differences were investigated. The results showed that 5μM Cd exposure caused a sharp decline(p≤0.05)in chlorophyll contents and net photosynthesis of the two barley genotypes with loosen chloroplast layer,and turgid grana and stroma lamellae in chloroplast were observed in both genotypes,granum stacking synchronously became incompact and some of them tend to degrade,furthermore,the deleterious effect of Cd was much severer in Cd-sensitive genotype Dong 17.Withal,increased accumulation in osmiophilic plastoglobuli and degradation of starch grain by Cd exposure were observed,and these deleterious effects were alleviated by GSH addition.Meanwhile,GSH supplement increased Pn,Tr and Sc of both genotypes under Cd stress.In addition,Cd stress impacted root ultrastructure,and resulted in ruleless sunken nuclear membrane,decline in the number and size of vacuole in root cells of both genotypes.Moreover,less number of cristae in mitochondria and plastids were found in the roots of Dong 17 under 5μM Cd,compared with control,while both of them remained unchanged and starch accumulated in plastids in Weisuobuzhi.Disservice in nucleus and mitochondria induced by Cd stress were almost recovered after GSH supplement.4.The effect of exogenous GSH on ROS metabolism in barley seedlings under Cd stress and genotypic differences was studied.The results showed that Cd stress induced the accumulation of O₂^?,H₂O₂ and MDA in leaves with genotypic difference,c.f.Dong 17 accumulated much more than Weisuobuzhi.The activities of antioxidant enzymes in Weisuobuzhi,particularly in roots,were significantly increased under Cd stress,while SOD,CAT and APX(especially cAPX)in the shoots of Dong 17 significantly decreased after 5-15 d Cd exposure.The accumulation of ROS and MDA was significantly reduced by GSH supplement.The induction of antioxidant enzymes such as SOD,CAT and APX activityies could be beneficial to decrease ROS and MDA accumulation to alleviate Cd induced oxidative stress.In Dong 17,CAT and APX activity were even higher than control after the addition of 20 mg L^-1GSH.POD and CAT responsed to Cd stress at expression level,but was not consistent with the trends of its activity.