Gene Expression Analysis Of Maize Seedling Root In Response To Heavy Metal Lead Stress

As the developing of agriculture and industry, the discharge of "the three wastes" and long run of chemical fertilizer and pesticide increase the content of heavy metals such as lead in the plough horizon, which not only restrict the normal growth and development of maize, but also endanger the health of animals and humans through the deliver of food chain. Maize is an important crop grown worldwide, what’s more, the stems and leaves are used to be the silage of livestock besides its seeds as food in some areas. With allocating a relatively higher proportion of its biomass in roots, maize could be a potential important model to study the phytoremediation of Pb-contaminated soil. Plants respond to survive under adverse conditions via a series of physiological, cellular, and molecular processes culminating in stress tolerance. However, little is known for its regulatory mechanism at epigenetic level and molecular mechanism in maize under Pb stress.In our previous research, we screened inbred lines of maize for Pb concentration, and line 178 was identified to be a hyperaccumulator for Pb in both the roots and aboveground parts, and the inbred 9782 was opposite. In this study, we aim to compare DNA methylation profiles in the dynamic development and transcriptome of inbred 9782 roots under Pb treatment to identify candidate genes for responding to Pb pollution. Here we analyzed the maize root transcriptome analysis of inbred lines 9782 under heavy metal lead (low-Pb) pollution, which was identified as a non-hyperaccumulator for low-Pb in roots. To identify important genes and metabolic pathways related to Pb hyperaccumulator, line 178 was underwent genome expression profile under Pb stress and a control (CK). These results will expand our understanding of the complex molecular and cellular events in maize root development and provide a foundation for future study on root development in maize under heavy metal pollution and other cereal crops. The main results were as follows:1. To understand the antioxidant metabolisms and lipid peroxidation in the responses of heavy metal tolerance in maize, a comparative physiology and biochemistry analysis of different maize genotypes (high-hyperaccumulator genotype 178, low-hyperaccumlator genotype 9782, and with the F1 generation of 178 and 9782) seedling roots exposed to 3 mM Pb(NO3)2 from o h to 192 h. As a result, the elongation growth of roots was significantly decreased under Pb stress and the number of hair roots was decreased too. The root growth traits of inbred 178 were weaker than inbred 9782 and hybrid F1 under Pb stress, but the relative activity of root both 178 and F1 were greater than 9782. The malondialdehyde (MDA) concent reflects the level of lipid peroxidation in maize under Pb stress. The MDA concentration of both 178 and Fl were lower than 9782 under Pb stress. In addition, only the weak cell wall was found on the root surface of 178, but both weak cell wall and something like neoplasm were discovered under Pb stress through scanning electron microscope (SEM). The lipid peroxidation caused by stress conditions can be scavenged and detoxified by SOD, POD and CAT protective enzyme. The superoxide dismutase (SOD) activity of 178 was significantly higher than 9782 and Fl. The peroxidase (POD) activity of 178 and F1 were significantly higher than 9782. Meanwhile, after suffering the Pb-stress by 96H, the catalase (CAT) activity of 178 was significantly higher than 9782 and F1.2. Methylated DNA immunoprecipitation-sequencing (MeDFP-seq) was used to investigate the genome-wide DNA methylation pattern in the maize roots. The results showed that the average methylation density was the highest in CpG islands (CGIs) followed by intergenic. Within the gene body, the methylation density of introns was higher than that of UTRs and exons. In total,3,857 methylated genes were found in four samples, including 1,805 differentially methylated genes of K2 Vs. A1,1,508 of K3 Vs. A1,1,660 of K4 Vs. A1. Further analysis showed that overall 140 genes exhibited altered DNA methylation in all three contrasts, which included some well-known stress responsive transcription factors and proteins, such as MYB, AP2/ERF, bZIP, Serine-threonine/tyrosine-protein, Pentatricopeptide repeat protein, RING zinc finger protein, F-box protein, Leucine-rich repeat protein as well as tetratricopeptide repeat protein.3. In the present study, more than 98 millions reads were mapped to define gene structure and detect polymorphism, thereby to qualify transcript abundance along roots development under Pb treatment of inbred 9782. A total of 17,707,17,440,16,998 and 16,586 genes were identified in maize roots at four developmental stages (0,12 h,24 h and 48 h) respectively and 2,825,2,626,2,161 and 2,260 stage-specifically expressed genes were also identified respectively. In addition, based on our RNA-Seqdata, transcriptomic changes during maize root development responsive to low-Pb were investigated. A total of 384 differentially expressed genes (DEGs) (log2Ratio≥1, FDR≤ 0.001) were identified, of which,36 genes with significant alteration in expression were detected in four developmental stages; 12 DEGs were randomly selected and successful validated by qRT-PCR. Interestingly, a large number of genes related to response to stimulus (45 DEGs), response to stresses (42 DEGs), especially protein catabolic-related genes (164 DEGs) and binding (224 DEGs) accumulated predominantly during maize root development. Additionally, many transcription factor families might act as the important regulators at different developmental stages, such as bZIP, ERF and GARP et al.4. A total of ～11 million cDNA tags were sequenced and 4,665,539 and 4,936,038 clean tags were obtained from the libraries of the test and CK, respectively. In comparison to CK, 2379 and 1832 genes were identified up-or down-regulated, respectively, more than five folds under Pb stress. Interestingly, all the genes were related to cellular processes and signaling, information storage and processing, or metabolism functions. Particularly, the genes involved in posttranslational modification, protein turnover, and chaperones; signal transduction, carbohydrate transport and metabolism; and lipid transport and metabolism significantly changed under the treatment. In addition, seven pathways including ribosome, photosynthesis, and carbon fixation were affected significantly, with 118,12,34,21,18,72 and 43 differentially expressed genes involved. The significant up-regulation of the ribosome pathway may reveal an important secret for Pb tolerance of line 178. And the sharp increase of laccase transcripts and metal ion transporters were suggested to account in part for Pb hyperaccumulation in the line.