Expression Pattern And Function Of MiR408 In Seed Development Of Rice(Oryza Sativa)

MicroRNAs (miRNAs) are-22 nt sequence-specific non-coding RNAs, which regulate target-gene expression at the posttranscriptional levels by direct cleavage of mRNAs or repression of mRNA translation. microRNA408 (miR408) is a conserved family that has been identified in many plants. Plantacyanins, copper/zinc superoxide dismutases and laccases are the conserved target genes of miR408. However, regulation mechanism and biological functions of the miR408 remain unknown in rice. Meanwhile, rice is an important food resource for human daily life and serves as a model species of monocotyledon plants. In the study herein, we focused on the expression pattern and regulation mechanism of Os-miR408, and analyzed the phenotypes of various transgenic lines to investigate the biological functions of miR408 on plant development. Our main results are presented as followings:1. Analysis of the expression pattern of Os-MIR408GUS staining of pMIR408:GUS reporter lines illustrated that MIR408 was highly expressed in embryo of seed at the stage of germination and maturation. RT-PCR analysis of precursor transcripts showed that the accumulation level of pre-miR408 decreased in embryos during seed germination. To be exact, it dropped sharply along with the depletion of endosperm.2. Validation of Os-miR408 effects on grain size and embryo deveopmentWe generated transgenic plants expressing the miR408 precursor, antisense miR408 and MIM408 under the control of the CaMV 35S promoter. T3 homozygous lines derived from several independent single-copy transformants were used for the further investigations. Compared with the wild-type, two main traits were observed in these transgenic lines. Firstly, some embryos of 35S:MIM408 transgenic lines abnormally developed, including a small proportion of double-embryo in a seed. Secondly, transgenic lines of 35S:MIM408 showed a significant decrease in grain width, numbers of second branch (25%) and grains (5.7%) per spike, together with a increase in numbers of first branch per spike, resulting in a reduction of grain weight (6%). Especially, width of white grain decreased about 12.5%in comparison with wild type. The most phenotypes of 35S:AsMIR408 were similar with those of 35S:MIM408. However, transgenic lines of 35S:MIR408 showed a slightly increase in grain weight.3. Characterization of a novel non-conserved target (VIN1) of Os-miR408The expression level of VIN1 in embryo was negative related with that of Os-miR408 in 35S:miR408 and 35S:MIM408 transgenic lines. We demonstrated that VIN1 (04g45290) was a target gene of Os-miR408 by bioinformatic analysis, transient assays in Nicotiana benthamiana and qRT-PCR detection. The main function of VIN1 is to catalyze sucrose into glucose and fructose, which plays great role in the development in the maturation and germinatation of the rice seeds, especially in the size of the seeds. Similar to MIR408, VIN1 was also highly expressed in embryo at the stage of seed germination and maturation. Furthermore, the grain sizes of 35S:VINa and 35S:VINb transgenic plants sharply declined in comparison to wild type. The decrease of grain width was 10.7%and 8.5%in brown grain of the two 35S:VIN1 transgenic lines, while 13.5%and 11.1%in white grain. Meanwhile, grain length also reduced 4%and 6.2%in brown grain, while 3.4%and 5.4%in white grain.4. Interaction of VIN1 and Os-miR408 regulated sugar content in seed developmentAnalyses of concentration of sugar showed that interaction of VIN1 and Os-miR408 affected sucrose content in seed development in rice. Compared with wild type, concentration of sucrose almost doubled in 35S:MIR408 transgenic lines at the stage of seed germination, while that of fructose and glucose kept stable. By contrast, concentration of sucrose dramatically dropped whereas that of fructose and glucose significantly increased in 35S:AsMIR408,35S:MIM408,35S:VINa and 35S:VINb transgenic plants. These results suggested a reverse relationship between Os-miR408 and OsVIN in sugar content during the seed development in rice.Taken together, our results show that miR408-mediated VIN1 expression regulates sugar contents and grain size in the seed development. It suggests that miR408/VIN1 module plays an important role in seed maturation in rice.