The Expression Pattern, Regulatory Mechanism And Biological Functions Of MicroRNA393 In Arabidopsis Thaliana | Posted on:2011-05-31 | Degree:Doctor | Type:Dissertation | Country:China | Candidate:Z H Chen | Full Text:PDF | GTID:1100330332983372 | Subject:Genetics | Abstract/Summary: | PDF Full Text Request | MicroRNAs (miRNAs) are endogenous 20~24 nucleotide RNAs which target complementary mRNA transcripts for cleavage or transcriptional repression. microRNA393 (miR393) is a conserved family that has been identified in many plants. In Arabidopsis, it is encoded by two loci, MIR393a and MIR393b, which generate two identical mature miRNAs. miR393 post-transcriptionally regulates mRNAs for the plant auxin receptor proteins TIR1 (Transport Inhibitor Response Protein 1) and three closely related proteins AFB1 (AUXIN SIGNALING F BOX PROTEIN 1),AFB2 and AFB3 in Arabidopsis. To date, the expression patterns, regulation mechanism and biological functions of the miR393-TIR1/AFBs module in auxin response and plant development are not fully understood. In the study herein, we focused on the expression pattern and regulation mechanism of AtMIR393 family members, and analyzed the phenotypes of different transgenic lines to investigate the biological functions of miR393 on auxin response and plant development.1 Analysis of the expression pattern of AtMIR3931.1 Small RNA Northern blot showed the spatial and temporal expression patterns of mature miR393. It was expressed abundantly in shoots and much less in roots in young seedlings, while strongly expressed in inflorescences, moderately in leaves and weakly in stem in flowering plants. RT-PCR analysis of precursor transcripts showed that MIR393a and MIR393b were not expressed in the same pattern, and the expression pattern of MIR393b was more similar to that of mature miR393. Meanwhile, the histochemical localization of GUS staining of pMIR393a:GUS and pMIR393b:GUS report lines indicated that MIR393a and MIR393b have similar spatial transcriptional patterns, and they both showed prominent expression in vascular bundles of primary root, hypocotyl, leaves, floral organs and siliques. Interestingly, GUS activity was never detected in root apical region or lateral root primordium for both MIR393a and MIR393b.1.2 Semi-quantitative RT-PCR of target mRNAs in different tissues did not show a clear opposite expression pattern between miR393 and target mRNAs. GUS staining of promoter:GUS transgenic lines revealed that there were at least two different types of expression relationships between miR393 and its targets including "co-expression" and "mutual exclusion". There existed a co-expression of MIR393 and their targets in most of the tissues we analyzed, such as shoot apical meristems, vascular bundles in leaves, central stele in hypocotyls and roots. Compared with their targets, the promoters of MIR393a and MIR393b were active in more restricted regions. Furthermore, the target genes were strongly expressed in root apical region or lateral root primordium where the expression of miR393 can not be detected.2 The regulatory mechanism of AtMIR393 transcription under phytohormone and abiotic stresses2.1 Northern blot assay showed that miR393 accumulated in response to exogenous IAA treatment, and qRT-PCR analysis of the precursor abundance showed that its induction was due to enhanced MIR393b transcription but not MIR393a. Real time RT-PCR of the TIR1/AFBs transcripts under IAA treatment did not show any remarkable changes, while the GUS activities of pTIRl/AFBs:GUS lines were elevated. It is probably that the posttranscriptional repression of TIR1 mediated by miR393 contributes to the homeostasis of TIR1 protein in response to high concentration of IAA treatment.2.2 qRT-PCR analysis showed that MIR393a transcription was induced by ABA and mannitol treatments, while MIR393b was induced by 2,4-D and cold. These data indicated that transcriptional regulation of specific miR393 locus was different in response to phytohormone and abiotic stresses.3 The possible functions of At-miR393 in auxin signaling pathway and plant normal development3.1 Transgenic 35S:MIR393a and 35S:MIR393b lines were obtained in order to investigate the role of miR393. Overexpression of miR393 resulted in decrease of the TIR1 F-box family mRNA levels except AFB1. Six mutations were introduced into the miR393 target site in the coding sequence and generated a miR393-resistant form of TIR1 (mTIR1). Both T3 homozygous 35S:mTIR1 and 35S:TIR1 transgenic lines were obtained and northern blot analysis revealed a further increase of TIR1 mRNA abundance in 35S:mTIR1 than 35S:TIR1. These data proved that TIR1, AFB2 and AFB3 were under negative regulation of miR393.3.2 Phenotypic analysis of the homozygous T3 plants showed that overexpression of miR393 led to longer primary root and hypocotyl, fewer lateral root numbers, which was in good accordance with the phenotypes of tir1-1 mutant. In contrast, overexpression of TIR1 increased lateral root numbers and overexpression of mTIR1 led to more dramatic changes as much shorter primary root and more lateral roots. These data demonstrated that the miR393-mediated regulation of TIR1 played essential roles in root system development, especially the lateral root initiation.3.3 The effect of exogenous IAA on root elongation domonstrated that overexpression of miR393 reduced the inhibition of primary root growth compared to the control, while mTIR1 overexpressing lines increased it. Real-time qRT-PCR analysis of three primary auxin-response genes DFL1/GH3.6 (At5g54510), AXR5/IAA1 (At4g14560) and MSG2/IAA19 (At3g 15440) showed that the miR393-mediated TIR1 regulation influenced the expression of these genes under control and auxin-treat conditions, which contributed to the alteration of plant auxin sensitivity.3.4 Ectopic overexpression mTIRl exhibited auxin-related pleiotropic phenotypes such as increased numbers of adventitious roots, extremely curled leaves with margins bending to the abaxial surface, upward petioles and fewer leaf numbers, dwarfish and strong apical dominance with only one stem and no branches, and even prolonged vegetative phase. These data suggested that precisely control of TIR1 abundance by miR393 was essential for plant auxin signaling which is required for normal plant development.3.5 Northern blot analysis was used to detect the miR393 accumulation in 35S:TIR1, 35S:mTIRl, tir1-1 and tirlafb quadruple mutants. The level of miR393 was elevated about 1.5-fold in 10-day-old 35S:mTIR1 seedlings, but not in 35S:TIR1. Conversely, it was reduced about 40% in tirl-1 and tirlafb quadruple mutants. These data demonstrated that miR393 was positively regulated by the level of TIR1.In summary, we propose a model for the regulation of auxin response by miR393/TIR1. Auxin triggers a transcriptional induction of AtMIR393b, leading to increased miR393 level. Transcriptional regulation of specific MIR393 locus also occurs in response to ABA and some abiotic stresses. The posttranscriptional repression of TIR1/AFBs genes by miR393 alters plant auxin responses. Meanwhile, high level of TIR1 promotes miR393 expression by a feedback loop. The specific regulation of MIR393 expression and the interaction between miR393 and its target indicates a fine adjustment to the role of miR393-TIR1 module, which is required for phytohormone and abiotic stress responses in plant development.
| Keywords/Search Tags: | microRNAs, Arabidopsis, miR393, target genes, TIR1/AFB gene family, lateral root development, auxin, sensitivity, expression pattern, function | PDF Full Text Request | Related items |
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