Regulation Of Target Gene Expression By AtMIR393Family And Its Role In Leaf Development

MicroRNAs （miRNAs） are endogenous 20～24 nucleotide RNAs which target complementary mRNA transcripts for cleavage or transcriptional repression. In leaf development, each cell in the primordium divides, grows and differentiates in a controlled way. Recent years, miRNAs have been found to be involved in the regulatory networks of leaf development. miRNA-mediated endogenous gene repression can specify the temporal-and spatial-expression pattern of their targets, and play crucial roles in leaf development.In Arabidopsis, microRNA393 （miR393） is encoded by two loci, MIR393a and MIR393b. AtmiR393 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 the study herein, we focused on the expression pattern of AtMIR393 family members, and analyzed the phenotypes of different transgenic lines to investigate the biological functions of miR393 in leaf development.1. Analysis of the expression pattern of AtMIR393 in leaf developmentThe histochemical localization of GUS staining of pMIR393a:GUS and pMIR393b:GUS report lines indicated that MIR393a and MIR393b have similar spatial transcriptional patterns, both in leaf vein, shoot apical meristem and hypocotyl. GUS staining has also been found in rosette leaves and stomatal apparatus. Interestingly, high accumulation of promoter activity was observed in the most distal region of young leaves, and the signal dispeared after another 1-week-growth. qRT-PCR analysis of miR393, MIR393 precursors and target mRNAs showed that the level of miR393 was much higher in old leaves （the 1st and 2nd pairs of leaves） than younger leaves（3rd and 4th pairs of leaves）, which was consistent with miR393 precursor levels. It indicated that transcriptional regulation of MIR393 might contribute to the dynamic pattern of miR393 during leaf development. Furthermore, the data of quantitative RT-PCR did not show a clear opposite expression pattern between miR393 and their target mRNAs.2. Generation of miR393 knock down transgenic plants In order to investigate the role of miR393, we generated various transgenic plants to knock down the function of miR393. These mutant lines included pTIR1:mTIRl （to express a miR393-resistant form of TIR1 （mTIRl））,35S:MIM393 （using artificial miRNA target mimics to negatively regulate the activity of miR393）,35S:anti393a and 35S:anti393b （to reveal the function differnence between MIR393a and MIR393b）. Furthermore, a T-DNA insersion mutant mir393a （SALK₁04430） has also been selected, and qRT-PCR showed that the level of MIR393a was decreased in mir393a. We also generated a miR393-GFP sensor constructs （35S.393 Sensor and 35S:393 muSensor） to elucidate the spatio-temporal basis of miRNA action during development.3. The possible functions of At-miR393 in leaf development 35S:cTIR1,35S:mTIR1 and pTIRl:mTIR plants exhibited auxin-related pleiotropic phenotypes such as extremely curled leaves with margins bending to the abaxial surface and upward petioles. Among these lines,35S:mTIR1 and pTIR1:mTIRl displayed more severe leaf curling and petiole upward, while the phenotypes of 35S.TIR1 and 35S:MIM393 were much milder.The statistics analysis of leaf traits suggested that the number of rosette leaves was positively related with the expression level of TIR1 gene. The number of rosette leaves increased in 35S:cTIR1,35S:mTIR1 and pTIRl:mTIR1, while it decreased in tir1-1. Moreover, the parameter of leaf epinasty increased in 35S:mTIR1 and pTIRl:mTIRl.We also measured the expression levels of other related genes in auxin signaling pathway and leaf development in these lines. GH3.3, which is one of primary auxin-response genes, was positively regulated by TIR1. It suggested that the regulation of TIR1 by miR393 plays an important role in leaf growth and development through auxin signaling pathway.