Kurdyukov Sergey, Mathesius Ulrike, Nolan Kim E, Sheahan Michael B, Goffard Nicolas, Carroll Bernard J, Rose Ray J
School of Environmental & Life Sciences, University of Newcastle, Callaghan, NSW, Australia.
BMC Plant Biol. 2014 Jun 21;14:174. doi: 10.1186/1471-2229-14-174.
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.
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.
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 3'UTR and is also associated with DNA methylation of MtEIL1. 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. The work also suggests that epigenetic changes to a particular gene induced in culture can be fixed in regenerated plants.
蒺藜苜蓿2HA种子系具有高度胚胎发生能力，而其亲本Jemalong很少产生胚胎。2HA系是从罕见的Jemalong再生植株之一培育而来，这种在蒺藜苜蓿中获得高度可再生基因型的方法易于重复，提示存在一种表观遗传机制。微阵列转录组分析显示2HA愈伤组织中一个乙烯不敏感3样基因表达下调，这为研究与乙烯信号传导相关基因的表观遗传调控及2HA表型提供了一种途径。乙烯参与包括体细胞胚胎发生（SE）在内的许多发育过程，并与应激反应相关。
微阵列转录组分析显示2HA组织培养中有大量上调转录本，包括根瘤和胚胎特异性基因以及转座子样基因，而只有少数基因下调，包括一个我们称为MtEIL1的EIN3样基因。这种表达降低与2HA植株的乙烯不敏感性有关，并对此进行了进一步研究。较弱的乙烯不敏感性影响根和根瘤发育。MtEIL1测序发现2HA与野生型植株之间没有差异。MtEIL1的DNA甲基化分析显示2HA与野生型植株之间存在显著差异。平铺阵列显示2HA植株中与MtEIL1基因反义链杂交的miRNA水平升高。AFLP样甲基化谱分析显示2HA与野生型之间在DNA甲基化方面存在更多差异。分离分析表明eil1表型为隐性，而SE性状为显性。
我们已经证明蒺藜苜蓿（MtEIL1）的EIL1在2HA种子系中发生了表观遗传沉默。可能的原因是靶向其3'UTR的miRNA水平升高，并且还与MtEIL1的DNA甲基化有关。MtEIL1的下调使得在有乙烯存在的情况下能够形成根瘤，并在正常条件下影响根的生长。分离分析表明MtEIL1表达与培养中的SE之间没有关联，但乙烯信号传导在通过SE进行植物再生过程中的作用和机制需要进一步研究。这项工作还表明，培养中诱导的特定基因的表观遗传变化可以在再生植株中固定下来。
