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Identifying genes involved in microRNA turnover in Arabidopsis

Periodic Reporting for period 1 - miRTurnOver (Identifying genes involved in microRNA turnover in Arabidopsis)

Reporting period: 2015-06-01 to 2017-05-31

The project with the acronym “miRTurnOver” aimed at the identification of genes involved in miRNA degradation or turnover in Arabidopsis. miRNAs are post-transcriptional regulators that control gene expression in plants, often responsive to environmental or developmental changes. A lot is known about how miRNA molecules are transcribed and produced from their precursors but we know little about how they are turned over once the plants do not need them anymore. It is important to understand how miRNAs are degraded because it is part of the plants’ response to environmental changes and as such it is important for plant breeding.
Using a system based on miR395, a sulfate deficiency induced miRNA, we screened EMS treated seeds of the transgenic SUC2::GFP-395 Arabidopsis line. This line expresses GFP in the phloem companion cells (from the SUC2 promoter) from a mRNA that carries a miR395 target site in its 3’prime untranslated region. When plants grow in low sulfate media MIR395 is switched on, GFP expression is blocked by miR395 but once sulfate is added to the media, miR395 transcription is switched off and the miRNA molecules already present will be turned over leading to GFP expression. We found that it takes a few days for this GFP recovery to occur in the non-mutant SUC2::GFP-395 plants (control). The scope of the screening was to identify putative EMS mutants that show GFP recovery earlier than the control and thus having accelerated miRNA degradation or later than the control and thus having slower miRNA degradation. Identification of these lines would allow us to discover what genes play a role in this pathway. We screened more than 13,000 EMS M2 seeds and identified 12 putative mutant plants. Seeds of these plants were planted to give the next generation (M3) that underwent the same screening. Until the end of the project, one putative mutant of early response (fast miRNA degradation) was further analysed and its fast response was confirmed in a time course experiment where the level of miR395 was measured by northern-blot during the recovery in the M3 generation. Plant tissue from M4 plants growing in the growth chamber along with control untreated SUC2::GFP-miR395 plants will be sent for RNA-sequencing in order to get an insight of the genes affected by the EMS and thus potentially involved in the fast degradation of the miRNAs observed in this mutant. This will inform us on how these small regulatory molecules that act as molecular switches are being degraded in Arabidopsis and will lay the foundations for the understanding of the degradation process in other plants.
Unexpected problems delayed the screen and initially more work was done to optimise the screen. Once that was completed we screened 13,200 seedlings (each was screened at 6 different time points, for details see technical report). Twelve mutant lines have been identified. Two of these were followed up where the level of miR396 was measured by northern blot in wild type and mutants plants in a time course experiment. The results of the project was displayed on a poster at a Small RNA conference.
We identified 12 mutants that display faster or delayed GFP recovery therefore miRNAs are potentially degraded faster or more slowly in these mutants. the impact is long term and indirect. Understanding how miRNAs are degraded may contribute to plant breeding programs to generate crops that are more tolerant to environmental changes.
measurment of miR395 level in wild type and 2 mutants during a time course experiment