Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of Medicago truncatula 2HA tissue culture with auxin treatment

ABSTRACT: Leaf explants of the superembryogenic Medicago truncatula line 2HA were treated with auxin (1-naphthaleneacetic acid) for one week to induce the formation of roots (Imin et al J Exp Bot 58:439-451). Gene expression in the leaves and the NAA treated tissue cultures was compared to identify transcripts expressed during the commitment to root formation in tissue culture. We have used the Affymetrix Medicago Genome Array GeneChip to compared gene expression in Medicago truncatula leaves and leaf explants that have been cultured for one week on NAA, to identify genes expressed during the commitment to root formation in tissue culture. Experiment Overall Design: Medicago truncatula 2HA leaves and leaf explants treated with NAA for one week were collected; total RNA was extracted and used for hybridization to Affymetrix arrays

ORGANISM(S): Medicago truncatula

SUBMITTER: Peta Holmes

PROVIDER: E-GEOD-8138 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: The Medicago truncatula 2HA seed line is highly embryogenic while the parental line Jemalong rarely produces embryos. The 2HA line was developed from one of the rare Jemalong regenerates and this method for obtaining a highly regenerable genotype in M. truncatula is readily reproducible suggesting an epigenetic mechanism. Microarray transcriptomic analysis showed down regulation of an ETHYLENE INSENSITIVE 3-like gene in 2HA callus which provided an approach to investigating epigenetic regulation of genes related to ethylene signalling and the 2HA phenotype. Ethylene is involved in many developmental processes including somatic embryogenesis (SE) and is associated with stress responses. Results: Microarray transcriptomic analysis showed a significant number of up-regulated transcripts in 2HA tissue culture, including nodule and embryo specific genes and transposon-like genes, while only a few genes were down-regulated, including an EIN3-like gene we called MtEIL1. This reduced expression was associated with ethylene insensitivity of 2HA plants that was further investigated. The weak ethylene insensitivity affected root and nodule development. Sequencing of MtEIL1 found no difference between 2HA and wild-type plants. DNA methylation analysis of MtEIL1 revealed significant difference between 2HA and wild-type plants. Tiling arrays demonstrated an elevated level of miRNA in 2HA plants that hybridised to the antisense strand of the MtEIL1 gene. AFLP-like methylation profiling revealed more differences in DNA methylation between 2HA and wild-type. Segregation analysis demonstrated the recessive nature of the eil1 phenotype and the dominant nature of the SE trait. Conclusions: We have demonstrated that EIL1 of Medicago truncatula (MtEIL1) is epigenetically silenced in the 2HA seed line. The possible cause is an elevated level of miRNA that targets its 3M-bM-^@M-^YUTR leading to DNA methylation. Down regulation of MtEIL1 makes it possible to form nodules in the presence of ethylene and affects root growth under normal conditions. Segregation analysis showed no association between MtEIL1 expression and SE in culture but the role and mechanism of ethylene signalling in the process of plant regeneration through SE requires further investigation. Comparison of transcriptome of WT and highly embryogenic 2HA spontaneous mutant of Medicago truncatula in 4 weeks tissue culture