The Regulation Role Of MiR396Mediated BASIC HELIX-LOOP-HELIX TRANSCRIPTION FACTOR74(bHLH74) Repression In Root Growth In Arabidopsis

In Arabidopsis thaliana, miR396family contains two members:miR396a and miR396b, which are encoded by MIR396a and MIR396b, respectively. miR396targets seven members of GRF (Growth-Regulating Factor) family and a basic Helix-Loop-Helix transcription factor bHLH74. Previous studies revealed that miR396-target regulates cell proliferation and plays a critical role in leaf development.In order to explore the function of different members of MIR396in root development, we studied the expression patterns of AtMIR396and its target bHLH74, and analyzed the root phenotype of T-DNA insertion mutants and different transgenic lines.1. The selection and phenotype observation of mir396a mutant.After two PCR cycles, homozygous mir396a-1mutant has been selected. Quantification RT-PCR detection indicated that the levels of both MIR396a precursor and mature miR396were down regulated. Phenotype observation showed that the primary root length of mir396a-1mutant seedlings were longer than that of wild-type. This indicated that miR396a was involved in root growth regulation in Arabidopsis.2. Function of the target gene bHLH74in root growth.GUS staining of probHLH74:GUS lines showed that the target gene bHLH74expressed both in the root and the leaves. It mainly expressed in the vascular bundle of primary root and the emerging lateral roots.35S:mbHLH74was constructed by introducing synonymous mutations in the coding sequence of bHLH74, which impaired its interaction with miR396. RT-PCR showed that the transcript levels of bHLH74in35S:mbHLH74transgenic lines were significantly increased. Furthermore, the primary root length was increased in the35S:mbHLH74, which was opposite to the phenotype of bhlh74-1. Root meristem of the seedlings was observed by using PI-stained root. The meristem cell number and meristem length were increased in both35S:mbHLH74transgenic plants and mir396a-1mutant, while decreased in bhlh74-1. It suggested that bHLH74, which targeted by miR396, can regulate root growth by affecting meristem size during root development.3. Analysis of sRNAs generated by MIR396a and MIR396b precursors.Small RNA sequencing of35S:MIR396a and35S:MIR396b transgenic plants, as well as examination of public small RNA deep sequencing data from Arabidopsis Col-0, showed that multiple distinct sRNAs originated from MIR396a and MIR396b precursors. To evaluate the efficiency of target cleavage, we co-transformed Nicotiana benthamiana leaves with different miR396and bHLH74constructs for transient expression. It showed that expression of miR396and its sRNA variants resulted in different efficiencies of target suppression. In addition, over expressing of MIR396a and MIR396b would affect the expression levels of other miRNA families. Those may be the reasons of the different phenotypes of35S:MIR396a and35S:MIR396b transgenic plants.