Identification Of Qtls And Candidate Genes Associated With Cadmium Tolerance And Accumulation In Barley And Its Regulation

Cadmium (Cd), a non-essential trace element, is one of the most phytotoxic heavy metals and widespread pollutant in the environment. Cd is easily absorbed and accumulated in plants and of toxicity at low concentrations, thus may induce potential human health risk via food chain. It is crucial important to understand the underlying mechanism associated with Cd tolerance and accumulation in plants, for developing Cd-resistant/tolerant and low accumulated cultivars or using agronomic practice to inhibit the involvement of Cd in food chains. The purpose of the present study is to elucidate physiological mechanism of genotypic difference in Cd uptake and distribution, using barley genotypes differing in Cd tolerance and accumulation, to identify specific proteins and relevant genes for Cd-tolerance/low accumulation. Meanwhile, genotypic difference were systemically investigated by employing the newly constructed suspension cell lines. With the aid of analyzing available SSR and SNP markers and the establishment/construction of genetic linkage map, Cd tolerance/accumulation relevant gene(s) were tagged/localized, and the genetic relationship was investigated between Cd accumulation and some morphological and physiological indexes, using DH population derived by F1hybrids of genotypes differing in Cd accumulation and tolerance. In addition, we investigated the possibility to reduce Cd uptake and accumulation in barley plants by application of exogenous N-acetyl cysteine (NAC) and its protective effect against Cd-induced growth inhibition, oxidative stress, and damage in ultrastructure and cell viability of roots tips in the two different genotypes differing in Cd tolerance. The main results were summarized follows:1. Genotypic differences in photosynthesis performance and Cd uptake and distribution in response to Cd toxicity in barley seedlingsA hydroponic experiment was carried out to examine genotypic differences in photosynthesis performance and Cd uptake and distribution in response to different Cd levels (0,5,50,500μM Cd), using two barley genotypes differing in Cd-tolerance and accumulation (c.f. W6nk2, Cd-sensitive and low-grain-accumulation; Zhenong8, Cd-tolerant and high accumulator). The results showed that plant height, root length, and biomass accumulation of shoot/root were significantly reduced in plants grown in5,50,500μM Cd compared with control. There was highly significant difference in the reduction of these growth parameters between the two genotypes, Zhenong8showed much less reduction, suggesting its high tolerance to Cd toxicity, while W6nk2with the greater reduction. Significant genotypic difference in Cd concentration was also found. Zhenong8contained higher Cd concentration in plant tissue than W6nk2. The results of Cd-specific fluorescence localization in root tip showed that Cd was mainly in apoplast, particularly within the cell wall, this phenomenon was more obvious in tolerant genotype Zhenong8. Within the same plant Cd concentration from the root tip to the shoot was gradually decreased, with the treatment time extended the Cd accumulation was higher, and there was a upward moving trend from the root tip to shoot. Speculating that the roots have the binding role to Cd spread, and therefore the roots were poisoned heavyly. Cd stress significantly affected photosynthesis and chlorophyll fluorescence of barley, and there were significant differences between different genotypes, Cd-sensitive genotype W6nk2severely inhibited, while Cd-tolerant genotype Zhenong8showed relatively strong resistance.2. Comparison study of grain protein expression between two different grain Cd accumulate genotypeWe compared grain protein expression in two barley genotypes differing in Cd tolerance and accumulation using two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF mass spcetrometry analysis. A total of29protein were identified differentially expression, of these alerted proteins,17were significantly higher expressed and12were significantly suppressed in Zhenong8compared with W6nk2grains. Overall, these differential expression protein were mainly on proteinase inhibitor, involving in higher expressed protein α-amylase/trypsin inhibitor CM, protein z-type serpin, serpin-Z7(HorvuZ7), and significantly suppressed protein BTI-CMe2.1protein, BTI-CMe2.2protein. Followed by stress-related protein, such as up-regultaed protein HSP70and RAB, member of RAS oncogene family-like4, suppressed protein dehydroascorbate reductase. There were also storage protein, embryo globulin and putative avenin-like a precursor were higher expressed in Zhenong8. It was indicated that these proteins might play an important role in Cd accumulation and tolerance or detoxification, and these protein identified may provide new opportunities for elucidating the barley Cd accumulation mechanism.3. Genetic linkage map construction of barley DH population and Cd tolerant related QTL mapping at seedling stageA mapping population of108lines (DH), derived from F1coming from a cross between Cd tolerant genotype Weisuobuzhi and Cd sensitive genotype Suyinmai2, were used in this investigation. The linkage map consisted of41SSR and15SNP markers-spanning396.9centimorgans (cM), with an average linkage distance between markers of8.1cM, and clustered into7molecular linkage groups (LGs). Hydroponic and pots experiments were carried out to study the barley growth differences and Cd tolerant related traits among different lines under Cd treatment during different develop stages, and mapped Cd related QTLs. Nine QTLs were detected, associated with Cd tolerance index (CTI) of SPAD value and growth traits on chromosomes1,2,5and7. The individual contribution of each QTL to the phenotypic variation was ranging3.30-9.49%. Twenty two QTLs controlling antioxidant enzymes and MDA content located on all seven chromosomes. Half of them were related in the shoots and half detected in the roots, with the average phenotypic variance5.6%. One pairs epistatic QTLs were detected, eg. Cd tolerance index (CTI) of root glutathione peroxidase (GPX) activity. In addition, four QTLs, which were the leaf numbers per plant, root CAT activity and root dry weight, dry weight at harvest were detected in the interval ABC2-Chitinase on2H, the position was0.0cM, suggesting that these QTLs maybe the same QTL and these traits were associated with ABC transporter. Under genetype and environment interaction conditions,6QTLs for Cd concentration/accumulation were mapped on chromosomes1,2,5,6,7. Two QTLs, qRCd1-1and qACd1-1, overlapped by the same genetic region of ABC1-GMS021, and2additive QTLs of qShCd2-3and qShCd7-2exist epistatic effect. There were7and11QTLs for microelement concentrations in shoots and roots, respectively, with the total contribution of16.37%and24.95%. The total phenotypic variation caused by environment interaction effect was12.41%and14.6%respectively.4. Analysis of QTLs associated with Cd accumulation and tolerance at different growth stagesIn pot experiment, eleven QTLs for Cd concentration with significantly additive (A) effect at different stages and in different organs were detected, some of them represented a single locus affecting different traits and/or the same trait under both control and Cd treatments. Two pairs epistatic QTLs were detected, i.e. one was kernel Cd accumulation with positive epistatic effect, being significant in additive×additive interactions by Cd environment interaction effects; the other was root Zn concentration related epistatic QTLs. In addition, one shoot Cd accumulation related QTL with significantly additive (A) effect and/or additive environment interaction (AE) effect was identified. For63microelements related QTLs,25and38were of leaves and different organs, respectively, and17of them with significant AE effect. Eight QTLs associated with CTI of agronomic and yield traits were detected with phenotypic variation1.54-10.79%. Furthermore, genetic regions EBmac0615-EBmatc0054, EBmac0557-EBmac0615and the novel SNP marker ABC transporters assigned to Cd and several microelement concentrations.5. Identification of relevant genes for Cd-tolerance and low accumulation, the candidate gene clone and transformation of high and low grain Cd accumulating barleyUsing cDNA microarray technology to analyze gene expression under5μM Cd stress and select Cd tolerant and low Cd accumulate related genes in two barley genotypes differing in Cd tolerance and accumulation. The result showed that there was significant difference on expression response of genes related to Cd stress between two genotypes, the up-regulated transcript levels of genes relating to metal transporters in plants were observed, such as ZIP, ABC transporter, P-type ATPase and iron-phytosiderophore transporter in leaves of W6nk2after Cd exposure, which might contribute to its low Cd accumulation characterization. Real time PCR result also confirmed these gene’s expression. Thus, we islolated some of them (e.g. ZIP family) from both barley genotypes and transformed them into Golden Promise, our hypothesis was that using RNAi technology may prove the low Cd accumulating gene by suppression of this gene expression in transgenic plants with elevated Cd concentration. 6. Establishment of suspension cells in different barley genotypes and analysis of its Cd toleranceHere the well-diffused and stable suspension cells were successfully constructed for the four barley genotypes differing in cadmium-tolerance and accumulation (Weisuobuzhi and Dong17were Cd tolerant and sensitive, Zhenong8and W6nk2were high and low Cd accumulate genotypes, respectively). The biological effects and the expression of protein ZIP7under Cd stress on four barley cells and the genotype difference were systemically investigated by the newly constructed suspension cell lines. The result showed that stable single suspension cells of four barley genotypes were successfully constructed using immature embryo with diameter2mm as explant. After50μM Cd treatment, the ratio of live suspension cells of four barley genotypes were decreased significantly compared with control. With increase in treating time, the survival of cells decreased, especially the Cd sensitive genotype Dong17and W6nk2. SDS-PAGE gel combined with Western blotting results indicated that the expression of protein ZIP7was upregulated after Cd treatment in W6nk2compared with Zhenong8, this result testified previous result by microarrays and RT-PCR.7. Effects of NAC application on Cd toxicity and its genotypic differenceA greenhouse hydroponic experiment was carried out, using two barley genotypes differing in Cd tolerance, to evaluate the alleviation effect of exogenous NAC against Cd-induced oxidative stress, barley growth inhibition, Cd and other nutrients uptake and ultrastructure damage. Addition of200μM NAC in50μM Cd culture medium significantly alleviated Cd-induced growth inhibition, Cd toxic symptoms was alleviated by NAC application, and the increases in plant height, root length and dry weight over its Cd treatment were much higher than that in Dong17. Meanwhile, exogenous NAC dramatically depressed ROS, MDA accumulation and antioxidant enzymes, compared with50μM Cd treatment, meliorated Cd-induced damages on leaf and root ultrastructure, the damage of the lamellar structure and shape of chloroplasts almost returned to the control level, a significant reduction in the number of osmiophilic plastolobuli, effectively increased antioxidant enzymes activity, improved the stability and integrity of the nuclear membrane of root meristem, and increased the cell viability in roots tips.