Laboratório de Biotecnologia de Células Vegetais, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
BMC Plant Biol. 2011 May 10;11:79. doi: 10.1186/1471-2229-11-79.
Small RNAs (sRNAs) are 20-24 nucleotide (nt) RNAs and are involved in plant development and response to abiotic stresses. Plants have several sRNA pathways implicated in the transcriptional and post-transcriptional silencing of gene expression. Two key enzyme families common to all pathways are the Dicer-like (DCL) proteins involved in sRNAs maturation and the Argonautes (AGOs) involved in the targeting and functional action of sRNAs. Post-transcriptional silencing mediated by AGOs may occur by cleavage or translational repression of target mRNA's, while transcriptional silencing may be controlled by DNA methylation and chromatin remodeling. Thus far, these gene families have not been characterized in legumes, nor has their involvement in adaptation to water deficit been studied.
A bioinformatic search in Medicago truncatula genome databases, using Arabidopsis thaliana AGO and DCL cDNA and protein sequences, identified three sequences encoding for putative Dicer-like genes and twelve sequences encoding for putative Argonaute genes. Under water deficit conditions and mainly in roots, MtDCL1 and MtAGO1, two enzymes probably involved in the processing and activation of microRNAs (miRNAs), increased their transcript levels. mir162 which target DCL1 mRNA and mir168 which target AGO1 mRNA reduced their expression in the roots of plants subjected to water deficit. Three putative genes, MtDCL3, MtAGO4b and MtAGO4c probably involved in DNA methylation mechanisms, increased their mRNA levels. However, the mRNA levels of MtAGO6 reduced, which probably encodes a protein with functions similar to MtAGO4. MtAGO7 mRNA levels increased and possibly encodes a protein involved in the production of trans-acting small interfering RNAs. The transcript abundance of MtAGO12a, MtAGO12b and MtAGO12c reduced under water deprivation. Plants recovered from water deprivation reacquire the mRNA levels of the controls.
Our work demonstrates that in M. truncatula the transcript accumulation of the components of small RNA pathways is being modulated under water deficit. This shows that the transcriptional and post-transcriptional control of gene expression mediated by sRNAs is probably involved in plant adaptation to abiotic environmental changes. In the future this will allow the manipulation of these pathways providing a more efficient response of legumes towards water shortage.
小 RNA（sRNA）是 20-24 个核苷酸（nt）的 RNA，参与植物发育和对非生物胁迫的反应。植物有几种 sRNA 途径参与基因表达的转录和转录后沉默。所有途径中共同的两个关键酶家族是参与 sRNA 成熟的 Dicer-like（DCL）蛋白和参与 sRNA 靶向和功能作用的 Argonautes（AGO）。AGO 介导的转录后沉默可能通过靶 mRNA 的切割或翻译抑制发生，而转录沉默可能受 DNA 甲基化和染色质重塑的控制。到目前为止，这些基因家族尚未在豆科植物中得到描述，也没有研究它们在适应水分亏缺中的作用。
使用拟南芥的 AGO 和 DCL cDNA 和蛋白质序列，在蒺藜苜蓿基因组数据库中进行生物信息学搜索，鉴定出三个编码推定 Dicer-like 基因的序列和十二个编码推定 Argonaute 基因的序列。在水分亏缺条件下，主要在根中，两个可能参与 microRNA（miRNA）加工和激活的酶 MtDCL1 和 MtAGO1，增加了它们的转录水平。miR162 靶向 DCL1mRNA，miR168 靶向 AGO1mRNA，在水分亏缺处理的植物根中降低了表达。三个推定基因 MtDCL3、MtAGO4b 和 MtAGO4c 可能参与 DNA 甲基化机制，增加了它们的 mRNA 水平。然而，编码可能具有与 MtAGO4 相似功能的蛋白质的 MtAGO6mRNA 水平降低。MtAGO7mRNA 水平增加，可能编码参与产生反式作用小干扰 RNA 的蛋白质。在水分剥夺下，MtAGO12a、MtAGO12b 和 MtAGO12c 的转录丰度降低。从水分剥夺中恢复的植物重新获得对照物的 mRNA 水平。
我们的工作表明，在蒺藜苜蓿中，在水分亏缺下，小 RNA 途径的组成部分的转录积累受到调节。这表明，sRNA 介导的基因表达的转录和转录后调控可能参与植物对非生物环境变化的适应。在未来，这将允许对这些途径进行操作，为豆科植物对水分短缺提供更有效的响应。
