Genome-wide association analysis of salinity responsive traits in Medicago truncatula.
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ABSTRACT:
SUBMITTER: Kang Y
PROVIDER: S-EPMC6850670 | biostudies-literature | 2019 May
REPOSITORIES: biostudies-literature
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Project description:DNA methylation has a potential role in controlling gene expression and may, therefore, contribute to salinity adaptation in plants. Caliph medic (Medicago truncatula) is a model legume of moderate salinity tolerance capacity; however, a base-resolution DNA methylome map is not yet available for this plant.In this report, a differential whole-genome bisulfite sequencing (WGBS) was carried out using DNA samples extracted from root tissues exposed to either control or saline conditions. Around 50 million differentially methylated sites (DMSs) were recognized, 7% of which were significantly (p?<?0.05, FDR?<?0.05) altered in response to salinity. This analysis showed that 77.0% of the contexts of DMSs were mCHH, while only 9.1% and 13.9% were mCHG and mCG, respectively. The average change in methylation level was increased in all sequence contexts, ranging from 3.8 to 10.2% due to salinity stress. However, collectively, the level of the DNA methylation in the gene body slightly decreased in response to salinity treatment. The global increase in DNA methylation due to salinity was confirmed by mass spectrometry analysis. Gene expression analysis using qPCR did not reveal a constant relationship between the level of mCG methylation and the transcription abundance of some genes of potential importance in salinity tolerance, such as the potassium channel KAT3, the vacuolar H+-pyrophosphatase (V-PPase), and the AP2/ERF and bZIP transcription factors, implying the involvement of other epigenetic gene expression controllers. Computational functional prediction of the annotated genes that embrace DMSs revealed the presence of enzymes with potential cellular functions in biological processes associated with salinity tolerance mechanisms.The information obtained from this study illustrates the effect of salinity on DNA methylation and shows how plants can remodel the landscape of 5-methylcytosine nucleotide (5-mC) in the DNA across gene structures, in response to salinity. This remodeling varies between gene regions and between 5-mC sequence contexts. The mCG has a vague impact on the expression levels of a few selected potentially important genes in salt tolerant mechanisms.
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Project description:BACKGROUND: MicroRNAs (miRNAs) are small, endogenous RNAs that play important regulatory roles in development and stress response in plants by negatively affecting gene expression post-transcriptionally. Identification of miRNAs at the global genome-level by high-throughout sequencing is essential to functionally characterize miRNAs in plants. Drought is one of the common environmental stresses limiting plant growth and development. To understand the role of miRNAs in response of plants to drought stress, drought-responsive miRNAs were identified by high-throughput sequencing in a legume model plant, Medicago truncatula. RESULTS: Two hundreds eighty three and 293 known miRNAs were identified from the control and drought stress libraries, respectively. In addition, 238 potential candidate miRNAs were identified, and among them 14 new miRNAs and 15 new members of known miRNA families whose complementary miRNA*s were also detected. Both high-throughput sequencing and RT-qPCR confirmed that 22 members of 4 miRNA families were up-regulated and 10 members of 6 miRNA families were down-regulated in response to drought stress. Among the 29 new miRNAs/new members of known miRNA families, 8 miRNAs were responsive to drought stress with both 4 miRNAs being up- and down-regulated, respectively. The known and predicted targets of the drought-responsive miRNAs were found to be involved in diverse cellular processes in plants, including development, transcription, protein degradation, detoxification, nutrient status and cross adaptation. CONCLUSIONS: We identified 32 known members of 10 miRNA families and 8 new miRNAs/new members of known miRNA families that were responsive to drought stress by high-throughput sequencing of small RNAs from M. truncatula. These findings are of importance for our understanding of the roles played by miRNAs in response of plants to abiotic stress in general and drought stress in particular.
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Project description:BackgroundThe Medicago truncatula (M. truncatula) line 2HA has a 500-fold greater capacity to regenerate plants in culture by somatic embryogenesis than its wild type progenitor Jemalong. To understand the molecular basis for the regeneration capacity of this super-embryogenic line 2HA, using Affymetrix GeneChip(R), we have compared transcriptomes of explant leaf cultures of these two lines that were grown on media containing the auxin NAA (1-naphthaleneacetic acid) and the cytokinin BAP (6-benzylaminopurine) for two weeks, an early time point for tissue culture proliferation.ResultsUsing Affymetrix GeneChip, GCRMA normalisation and statistical analysis, we have shown that more than 196 and 49 probe sets were significantly (p < 0.05) up- or down-regulated respectively more than 2 fold in expression. We have utilised GeneBins, a database for classifying gene expression data to distinguish differentially displayed pathways among these two cultures which showed changes in number of biochemical pathways including carbon and flavonoid biosynthesis, phytohormone biosynthesis and signalling. The up-regulated genes in the embryogenic 2HA culture included nodulins, transporters, regulatory genes, embryogenesis related arabinogalactans and genes involved in redox homeostasis, the transition from vegetative growth to reproductive growth and cytokinin signalling. Down-regulated genes included protease inhibitors, wound-induced proteins, and genes involved in biosynthesis and signalling of phytohormones auxin, gibberellin and ethylene. These changes indicate essential differences between the super-embryogenic line 2HA and Jemalong not only in many aspects of biochemical pathways but also in their response to auxin and cytokinin. To validate the GeneChip results, we used quantitative real-time RT-PCR to examine the expression of the genes up-regulated in 2HA such as transposase, RNA-directed DNA polymerase, glycoside hydrolase, RESPONSE REGULATOR 10, AGAMOUS-LIKE 20, flower promoting factor 1, nodulin 3, fasciclin and lipoxygenase, and a down-regulated gene ETHYLENE INSENSITIVE 3, all of which positively correlated with the microarray data.ConclusionWe have described the differences in transcriptomes between the M. truncatula super-embryogenic line 2HA and its non-embryogenic progenitor Jemalong at an early time point. This data will facilitate the mapping of regulatory and metabolic networks involved in the gaining totipotency and regeneration capacity in M. truncatula and provides candidate genes for functional analysis.
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Project description:Heavy metals are an increasing problem due to contamination from human sources that and can enter the food chain by being taken up by plants. Understanding the genetic basis of accumulation and tolerance in plants is important for reducing the uptake of toxic metals in crops and crop relatives, as well as for removing heavy metals from soils by means of phytoremediation. Following exposure of Medicago truncatula seedlings to cadmium (Cd) and mercury (Hg), we conducted a genome-wide association study using relative root growth (RRG) and leaf accumulation measurements. Cd and Hg accumulation and RRG had heritability ranging 0.44 - 0.72 indicating high genetic diversity for these traits. The Cd and Hg trait associations were broadly distributed throughout the genome, indicated the traits are polygenic and involve several quantitative loci. For all traits, candidate genes included several membrane associated ATP-binding cassette transporters, P-type ATPase transporters, oxidative stress response genes, and stress related UDP-glycosyltransferases. The P-type ATPase transporters and ATP-binding cassette protein-families have roles in vacuole transport of heavy metals, and our findings support their wide use in physiological plant responses to heavy metals and abiotic stresses. We also found associations between Cd RRG with the genes CAX3 and PDR3, two linked adjacent genes, and leaf accumulation of Hg associated with the genes NRAMP6 and CAX9. When plant genotypes with the most extreme phenotypes were compared, we found significant divergence in genomic regions using population genomics methods that contained metal transport and stress response gene ontologies. Several of these genomic regions show high linkage disequilibrium (LD) among candidate genes suggesting they have evolved together. Minor allele frequency (MAF) and effect size of the most significant SNPs was negatively correlated with large effect alleles being most rare. This is consistent with purifying selection against alleles that increase toxicity and abiotic stress. Conversely, the alleles with large affect that had higher frequencies that were associated with the exclusion of Cd and Hg. Overall, macroevolutionary conservation of heavy metal and stress response genes is important for improvement of forage crops by harnessing wild genetic variants in gene banks such as the Medicago HapMap collection.
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Project description:Polycomb group (PcG) proteins, which are important epigenetic regulators, play essential roles in the regulatory networks involved in plant growth, development, and environmental stress responses. Currently, as far as we know, no comprehensive and systematic study has been carried out on the PcG family in Medicago truncatula. In the present study, we identified 64 PcG genes with distinct gene structures from the M. truncatula genome. All of the PcG genes were distributed unevenly over eight chromosomes, of which 26 genes underwent gene duplication. The prediction of protein interaction network indicated that 34 M. truncatula PcG proteins exhibited protein-protein interactions, and MtMSI1;4 and MtVRN2 had the largest number of protein-protein interactions. Based on phylogenetic analysis, we divided 375 PcG proteins from 27 species into three groups and nine subgroups. Group I and Group III were composed of five components from the PRC1 complex, and Group II was composed of four components from the PRC2 complex. Additionally, we found that seven PcG proteins in M. truncatula were closely related to the corresponding proteins of Cicer arietinum. Syntenic analysis revealed that PcG proteins had evolved more conservatively in dicots than in monocots. M. truncatula had the most collinearity relationships with Glycine max (36 genes), while collinearity with three monocots was rare (eight genes). The analysis of various types of expression data suggested that PcG genes were involved in the regulation and response process of M. truncatula in multiple developmental stages, in different tissues, and for various environmental stimuli. Meanwhile, many differentially expressed genes (DEGs) were identified in the RNA-seq data, which had potential research value in further studies on gene function verification. These findings provide novel and detailed information on the M. truncatula PcG family, and in the future it would be helpful to carry out related research on the PcG family in other legumes.
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Project description:Cytochrome P450 monooxygenases (P450s) catalyze a great number of biochemical reactions and play vital roles in plant growth, development and secondary metabolism. As yet, the genome-scale investigation on P450s is still lacking in the model legume Medicago truncatula. In particular, whether and how many MtP450s are involved in drought and salt stresses for Medicago growth, development and yield remain unclear. In this study, a total of 346 MtP450 genes were identified and classified into 10 clans containing 48 families. Among them, sixty-one MtP450 genes pairs are tandem duplication events and 10 MtP450 genes are segmental duplication events. MtP450 genes within one family exhibit high conservation and specificity in intron-exon structure. Meanwhile, many Mt450 genes displayed tissue-specific expression pattern in various tissues. Specifically, the expression pattern of 204 Mt450 genes under drought/NaCl treatments were analyzed by using the weighted correlation network analysis (WGCNA). Among them, eight genes (CYP72A59v1, CYP74B4, CYP71AU56, CYP81E9, CYP71A31, CYP704G6, CYP76Y14, and CYP78A126), and six genes (CYP83D3, CYP76F70, CYP72A66, CYP76E1, CYP74C12, and CYP94A52) were found to be hub genes under drought/NaCl treatments, respectively. The expression levels of these selected hub genes could be induced, respectively, by drought/NaCl treatments, as validated by qPCR analyses, and most of these genes are involved in the secondary metabolism and fatty acid pathways. The genome-wide identification and co-expression analyses of M. truncatulaP450 superfamily genes established a gene atlas for a deep and systematic investigation of P450 genes in M. truncatula, and the selected drought-/salt-responsive genes could be utilized for further functional characterization and molecular breeding for resistance in legume crops.
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Project description:Auxin response factors (ARFs) bind specifically to auxin response elements (AuxREs) in the promoters of down-stream target genes and play roles in plant responses to diverse environmental factors. Using the latest updated Medicago truncatula reference genome sequence, a comprehensive characterization and analysis of 24 MtARF (M. truncatula ARF) genes were performed. To uncover the basic information and functions of MtARF genes during symbiosis, we analyzed the expression patterns of MtARF genes during the early phase of Sinorhizobium meliloti infection. The systematic analysis indicated that changes in MtARF gene expression occur during these early stages of infection, suggesting a functional role in symbiosis. Furthermore, the roles of MtARF-mediated auxin signaling in symbiosis were tested in the infection resistant mutant (dmi3). The expression responses of MtARFs to S. meliloti infection were attenuated in the mutant compared to wild-type A17. In summary, our results show that changes in MtARF gene expression occur during the response to S. meliloti infection, suggesting that members of this family may have important roles in the symbiotic interaction.
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Project description:Numerous studies have shown that plant microRNAs (miRNAs) play key roles in plant growth and development, as well as in response to biotic and abiotic stresses; however, the role of miRNA in legumes under aluminum (Al) stress have rarely been reported. Therefore, here, we aimed to investigate the role of miRNAs in and their mechanism of Al tolerance in legumes. To this end, we sequenced a 12-strand-specific library of Medicago truncatula under Al stress. A total of 195.80 M clean reads were obtained, and 876 miRNAs were identified, of which, 673 were known miRNAs and 203 were unknown. A total of 55 miRNAs and their corresponding 2,502 target genes were differentially expressed at various time points during Al stress. Further analysis revealed that mtr-miR156g-3p was the only miRNA that was significantly upregulated at all time points under Al stress and could directly regulate the expression of genes associated with root cell growth. Three miRNAs, novel_miR_135, novel_miR_182, and novel_miR_36, simultaneously regulated the expression of four Al-tolerant transcription factors, GRAS, MYB, WRKY, and bHLH, at an early stage of Al stress, indicating a response to Al stress. In addition, legume-specific miR2119 and miR5213 were involved in the tolerance mechanism to Al stress by regulating F-box proteins that have protective effects against stress. Our results contribute to an improved understanding of the role of miRNAs in Al stress in legumes and provide a basis for studying the molecular mechanisms of Al stress regulation.
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