Physiological And Molecular Mechanism Of Cadmium Uptake And Translocation In Barley

Cadmium (Cd) is one of the most deleterious heavy metals to both plants and animalsand has no beneficial biological function in the aquatic or terrestrial organism, but can beabsorbed and accumulated easily by plants, while high accumulation of Cd in plants notonly affects crop yield and quality badly, but also gives rise to a threat on human health viafood 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 asselection/breeding of crop genotypes/cultivars tolerant to Cd toxicity and with low Cdaccumulation in edible parts, the improvement of agronomic practice and application ofchemical regulators which can reduce plant Cd uptake would be a cost-effective andpractical substitute mode to fully utilize natural resource and guarantee safe foodproduction. Accordingly, the present study was carried out to elucidate the physiologicaland molecular mechanism of Cd uptake and translocation in barley, and to identify specificproteins and relevant genes for Cd- tolerance/low accumulation through proteome andgenome analysis, based on the selection of genotypes differing in Cd accumulation andtolerance. Meanwhile, we investigated the possibility to reduce Cd uptake andaccumulation in barley plants by application of exogenous nitric oxide (NO) andglutathione (GSH) and the protective effect of exogenous NO against Cd-induced growthinhibition, oxidative stress, and damage in ultrastructure and photosynthesis in the twoselected genotypes differing in Cd tolerance. The main results were summarized follows:1. Identification of barley genotypes with low grain Cd accumulation and tolerance toCd toxicity.The variation in grain Cd concentrations was evaluated among 600 barley genotypesgrown in the same field condition to select low Cd accumulating genotypes. The resultsshowed that there is considerable genotypic variation in grain Cd concentrations in barleygrain samples, with the mean concentration of 0.16 mg kg^-1 DW and the variation of 0 (notdetected) to 1.21 mg kg^-1 DW, and 47.2% of the grain samples exceeded the maximumpermissible concentration (MPC) for Cd in cereal grains. In addition, differences betweengenotypes over the two years were fairly consistent, and Beitalys and Shang 98-128 showedthe lowest grain Cd concentration, being 97.5% lower than that in the two highest Cdaccumulators E-barley 6 and Zhenong 8 in the second harvest year. The great genotypic differences in Cd concentrations indicated that it is possible to lower Cd content of barleythrough cultivar selection and breeding for use at sites where Cd concentration in grainexceeds the MPC. Significant genotypic difference was also found in microelementconcentrations. Correlation analysis showed that only Mn accumulation is synergetic withCd accumulation, despite slightly positive relationship between Cd and Zn, Cu, or Fe inaccumulation in barley grains.Two successive hydroponic experments were carried out to identify barley varietiestolerant to Cd toxicity via examing SPAD value, dry weight, plant height, root length andvolume, tillers per plant, and biomass accumulation. The results showed that SPAD value,dry weight, plant height, root length and volume, and biomass accumulation weresignificantly reduced in the plants grown in 20μM Cd compared with control. There was ahighly significant difference in the decline of these growth parameters among genotypes.Weisuobuzhi and Jipi 1 showed the least reduction in both experiments, suggesting theirhigh tolerance to Cd toxicity, while Dong 17 and Suyinmai 2 with the greatest decline andtoxicity symptoms appeared rapidly and severely, donoting as Cd-sensitive genotypes. Inaddition, significant genotype difference in Cd concentration was also found, withWeisuobuzhi containing the highest, and Dong 17 had lower Cd concentration comparedwith Suyinmai 2.2. Cadmium transiocation and accumulation in developing barley grains and asaffected by some environmental factors.In order to study the transport of Cd into the developing grains, detached ears oftwo-rowed barley cv. ZAU 3 were cultured in Cd stressed nutrient solution containing themarkers for phloem (rubidium) and xylem (strontium) transport. Cd concentration in eachpart of detached spikes increased with external Cd levels, and Cd concentration in variousorgans over the three Cd levels of 0.5, 2, 8 M Cd on 15-day Cd exposure was in the order:awn > stem > grain > rachis > glume, while the majority of Cd was accumulated in grainswith the proportion of 51.0% relative to the total Cd amount in the five parts of detachedspikes. Cd accumulation in grains increased not only with external Cd levels but the time ofexposure contrast to stem, awn, rachis and glume. Those four parts of detached spikeshowed increase Cd accumulation for 5 days, followed by sharp decrease till day 10 andincrease again after 12.5 days. Awn-removal and stem-girdling markedly decreased Cdconcentration in grains, and sucrose or zinc (Zn) addition to the medium and higher relativehumidity (RH) also induced dramatic reduction in Cd transport to developing grains. Theresults indicated that awn, rachis and glume may involve in Cd transport into developing grains, and suggested that Cd redistribution in maturing cereals be considered as animportant physiological process influencing the quality of harvested grains. Our resultssuggested that increasing RH to 90% and Zn addition in the medium at grain filling stagewould be beneficial to decrease Cd accumulation in grains.3. Genotypic difference of Cd transporting into the developing grains.Genotypic difference of Cd transport into the developing grains was investigated usingdetached ears of two barley genotypes cultured in nutrient solution containing 0.5 and 5μMCd. The results showed that Cd concentration in each part of ears in W6nk2 (low grain Cdaccumulation genotype) was extremely lower than these in Zhenong 8 (high accumulator)in 0.5μM Cd treatment. However, grain Cd concentration of W6nk2 increased obviouslywith external Cd levels, and even reached the level of Zhenong 8 in 5μM Cd treatment.Awn-removal, high relative humidity (RH) and sucrose addition markedly decreased Cdconcentration in grains of Zhenong 8. but affected slighter in Cd transport to the grains ofW6nk2. Stem girdling also reduced the Cd transport to developing grains in 5μM Cdtreatment, especially for the low kernel Cd accumulation genotype W6nk2, while no effecton Cd transport to developing grains of both genotypes in low Cd treatment. Our resultssuggested that the different ability of Cd translocation in the xylem and phloem sap mightcontribute to the difference in grain Cd accumulation of the two genotypes. The results alsosuggest that screening the cultivar with low xylem transport, and regulated the phloemtransport at grain filling stage would be beneficial to decrease Cd accumulation in grains.4. Identification of specific proteins and relevant genes for Cd- tolerance/lowaccumulation.To begin to unravel the as-yet poorly understood molecular mechanisms of Cd uptake,translocation and tolerance in barley plants, we compared gene expression in four barleygenotypes differing in Cd tolerance and accumulation using cDNA microarrays. BarleylAffymetrix GeneChip was used to analyze the transcriptional response of barley plantsexposure to 0 (control) and 5μM Cd. The results showed that there was significantdifference on expression response of genes related to Cd stress among four genotypes.Transcriptional levels of 514 and 658 genes in low Cd accumulation genotype W6nk2 andCd sensitive genotype Dong 17 altered after plants exposed to 5μM Cd for 15 d, while only413 and 305 genes altered in Zhenong 8 (high Cd accumulation genotype) and Weisuobuzhi(Cd tolerant genotype) under Cd stress. The up-regulated transcript levels of genes relatingto metal transporters in plants were observed, such as ABC transporter, P-type ATPase, iron-phytosiderophore transporter and zinc transporter protein ZIP1 in leaves of W6nk2after Cd exposure, which might contribute to its low Cd accumulation characterization.Compared to Cd tolerant genotype Weisuobuzhi, more metal transport, photosynthesis andsignal transduction related genes were detected in Cd sensitive genotype Dong 17 under Cdstress. Meanwhile, the transcript level of PCS was not changed in Weisuobuzhi butdown-regulated in Dong 17, in addition, some antioxidant enzymes (eg. CAT) wereup-regulated in Weisuobuzhi, indicating that increasing synthesize of PCs and the activityof antioxidant enzymes were important to Cd tolerance in barley plants. In the other hand,proteomic analysis was used to investigate the Cd stress-responsive proteins in differentbarley genotypes, significantly genotypic difference on expression response of proteinsrelated to Cd stress was also found in two barley genotypes. Mass spectrometry analysisand database searching helped us to identify 28 spots representing 17 different proteins,involving in regulation of antioxidant enzymes, signal transduction, metal transport andphotosynthesis. Two of them (c.f. H^?-transporting two-sector ATPase and putative glycinedecarboxylase subunit) were up-regulated in Weisuobuzhi but not changed in Dong 17,indicating that these two proteins might play an important role in Cd tolerance.5. Effect of external NO and GSH supply on plant growth and microelementsaccumulation of two barley genotypes under Cd stress.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), 5μM Cd+0.25mM SNP (Cd+NO), and 5μM Cd+20 mg L^-1 GSH (Cd+GSH),to study the genotypic difference in the effect of exogenous NO and GSH on barleygrowth, Cd and other nutrients uptake and translocation under Cd stress. The resultsindicated that addition of 20mgL^-1 GSH (Cd+GSH) significantly reduced Cd uptake andaccumulation compared with Cd treatment and being emphasized in Dong 17. While0.25mM SNP added (Cd+NO) only reduced the concentration of Cd, but increased theaccumulation in roots of both genotypes, especially in Dong 17. Cadmium toxic symptomswas alleviated even more significantly in Dong 17 by NO and GSH application, and theincreases in plant height, root length and dry weight over its Cd treatment were muchhigher than that in Weisuobuzhi. It could be concluded that reduced Cd concentration andaccumulation in plants could be one of principal protective mechanism for the exogenousGSH in cytoprotection against Cd toxicity. GSH would be suitable for the edible cropsgrown in Cd soils to reduce Cd accumulation and alleviate phyto-toxicity of Cd. 6. Mechanism of alleviation effects of NO application on Cd toxicity and its genotypicdifference.A greenhouse hydroponic experiment was carried out, using two barley genotypesdiffering in Cd tolerance, to evaluate the protective effect of exogenous NO againstCd-induced oxidative stress, and ultrastructure and photosynthesis damage. Addition of0.25 mM SNP in 5μM Cd culture medium significantly alleviated Cd-induced growthinhibition, and its beneficial effect was observed in all growth parameters especially for thesensitive genotype Dong 17. Exogenous NO dramatically depressed ROS and MDAaccumulation, compared with 5μM Cd treatment, meliorated Cd-induced damages on leafand root ultrastructure, and increased chlorophyll content, Pn, Gs and Tr, thus improvedphotosynthesis efficiency. Meanwhile, exogenous NO counteracted Cd-induced time- andgenotype- dependent response of antioxidant enzymes, via suppressing the Cd-induceddramatic increase of POD activities in shoots of both genotypes recovery to near the controlvalues, and by elevating depressed APX, and CAT activities in Dong 17 after 10, and 15 dtreatment. The examination of APX and SOD isoenzymes in leaves revealed NOsignificantly increased sAPX and MnSOD activities in the both genotypes, and stronglystimulated Cd-induced decrease in cAPX (the major isoenzyme in barley leaves) insensitive genotype, but down-regulated the increased level in Weisuobuzhi. The results ofRT-PCR showed that POD, CAT1 and cAPX responded to Cd stress at transcript level.External NO up-regulated root and leaf cAPX and leaf CAT1 expression in Dong 17 toachieve stimulation. It could be concluded that improved photosynthesis efficiency and themembrane-stabilizing/integrity effect could be principal protective mechanism for theexogenous NO in cytoprotection against Cd toxicity. The results also suggested a practicalpotential for NO as a potent antioxidant in plants and that its action may, at least in part, beexplained by its ability to directly and indirectly scavenge ROS.