| The problem of heavy metal pollution is becoming a major worldwide ecological concern due to its potential adverse impacts on human health through the food chain. Lead (Pb) is one of the most toxic heavy metals and can be easily taken up by plants and accumulated in different parts. Radish{Raphamis sativus L.), is an economically important vegetable crop with an edible taproot. Because the root was considered as the vulnerable part that is affected by the heavy metal Pb, it has become of vital importance to investigate the Pb response mechanisms and explore the regulatory network of tolerance and homeostasis in radishes. Considerable efforts have been invested into investigating Pb stress in radishes, especially its accumulation, translocation, physiological and metabolic variations. However, little is known about the mechanism of radish in response to Pb stress at the molecular level. It is a fundamental step to isolate the functional-genes,proteins and metabolites for revealing the molecular mechanisms invlolved in reponse to Pb stress of radish. In this study, Next-Generation Sequencing (NGS)-based RNA-sequencing (RNA-seq) technology was employed to characterize the de novo transcriptome of radish roots taking an advanced inbred line ’NAU-RG’as the plant material, and the regulatory networks at different molecular levels including transcriptomic, proteomic and metabolomic studies in response to Pb stress of radish roots were revealed. The main achievements obtained were as follows.1. To develop a comprehensive overview of the radish root transcriptome, a cDNA library named’CKA’, prepared from three mixed RNA of taproots at different developmental stages including seedling, taproot thickening, and mature stages was subjected to pair-end read (PE) sequencing with the Solexa Illumina platform. Approximately 66.11 million paired-end clean reads representing 73,084 unigenes with a N50 length of 1,095 bp, and a total length of 55.73 Mb were obtained. A total of 67,305 (about 92.09%of the assembled unigenes) unigenes were successfully annotated using the publically available protein database. The functional annotation and classification including NCBI non-redundant protein (Nr), Gene Ontology (GO), Clusters of Orthologous Group (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the main activated genes of radish taproots were predominately involved in basic physiological and metabolic processes, biosynthesis of secondary metabolite pathways, signal transduction mechanisms and other cellular components and molecular function related terms. Our transcriptome dataset will serve as an important public information platform to improve the understanding of molecular mechanisms involved in taproot formation and the responses to various stresses.2. To isolate the differentially-regulated genes involved in Pb stress networks at mRNA level, two RNA libraries of untreated (CK) and Pb-treated (Pb10000) roots were constructed and sequenced using RNA-Seq approach. A total of 4,614 significantly differentially-expressed genes (DEGs) were detected consisted of 2,154 up- and 2,460 down-regulated genes between the two libraries. GO and KEGG pathway enrichment analysis revealed that upregulated DEGs under Pb stress were predominately involved in defense responses in cell walls and glutathione metabolism-related processes, while downregulated DEGs were mainly involved in carbohydrate metabolism-related pathways. The expression patterns of 22 selected genes were validated by qRT-PCR, and the results were highly accordant with the Solexa analysis. Furthermore, many candidate genes, which were involved in defense and detoxification mechanisms including signaling protein kinases, transcription factors, metal transporters and chelate compound biosynthesis related enzymes, were successfully identified in response to Pb stress of radish.3. To systematically isolate and dissect Pb-responsive miRNAs and their targets at the global level in radish roots, we constructed two small RNA libraries from one untreated control (CK) and one Pb-stressed (Pb500) of radish roots. Using Solexa sequencing technology, a total of 74 known and 173 novel candidate miRNAs were successfully identified from two libraries. Of these,25 known and nine novel miRNAs were significantly differentially expressed and identified as Pb-responsive miRNAs. Based on the reference sequences,1,979 miRNA-mRNA target transcripts could potentially be cleaved with degradome analysis. GO analysis revealed that these target transcripts were predominately involved in the regulation of transcription, defense responses, and binding related terms. The identified target genes for Pb-responsive miRNAs were mainly involved in stress-related signal sensing and transduction, specific metal uptake and homeostasis mechanisms.4. The changes in the protein profile of radish roots were comprehensively analyzed using iTRAQ (Isobaric Tag for Relative and Absolute Quantification) respectively upon Pb500 and Pb1000 exposure. A total of 3,898 proteins were successfully detected at a 95% confidence limit, and 2,141 were reliably quantified. A subset of 721 proteins was differently regulated upon at least one Pb treatment, and 135 proteins showed abundance changes during both two Pb-stressed conditions. GO and KEGG pathway enrichment analysis revealed that the 135 co-differently expressed proteins were strongly enriched in the categories of cell structure, carbohydrate and energy metabolism-related pathways and antioxidative defense. Furthermore, the changes of protein levels were compared with transcript levels of radish in response to Pb stress. Although the transcripts could be detected 77.3%and 77.9% respectively for the identified and quantified proteins, the abundance ratios showed large discrepancies between the levels of mRNA and protein expression in radish roots response to Pb stress.5. The metabolite profiling analysis of radish roots exposed to Pb stress has been performed using gas chromatography-mass spectrometry (GC-MS). The score plots of principal component analysis (PCA) and partial least squares-discriminate analysis (PLS-DA) showed clear discrimination between control and Pb-treated samples. The altered metabolites were found from the line plots of the X-loadings of the first component of the PLS-DA pairwise comparison models. On the basis of the parameter VIP>1 using student’s T test (T-test),28 Pb-metabolites with significant changes were identified (P<0.05), which mainly on saccharides, amino acids, and organic acids. Furthermore, an integrated analysis of the effects of Pb stress was performed on the levels of metabolites and gene transcripts in radish roots, and the integrative biochemical networks of the radish in response to Pb stress was characterized. KEGG pathway analysis of integration data demonstrated that exposure of radish to Pb stress resulted in profound biochemical changes including carbohydrate metabolism, energy metabolism, and glutathione metabolism. |
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