Study : FPS down-regulation alters chloroplast development and triggers an sterol-dependent induction of JA-related and Fe deficiency transcriptional responses
FPS down-regulation alters chloroplast development and triggers an sterol-dependent induction of JA-related and Fe deficiency transcriptional responses
In order to study the role of Farnesyl diphosphate synthase (FPS) in post-embryonic plant development, we generated Arabidopsis conditional knockdown mutants expressing amiRNAs devised to simultaneously silence both FPS (FPS1 and FPS2) genes. The expression of the amiRNAs is regulated by using a methoxyfenozide (MFZ) inducible promoter. An RNAseq analysis was made to compare the differentially expressed genes between control (not induced) and silenced (MFZ-induced) amiFPSa Arabidopsis seedlings. Differential gene expression analysis shows that a reduction in FPS activity levels triggers misregulation of genes involved in biotic and abiotic stress responses, the most prominent being the rapid induction of a set of genes related to the jasmonic acid (JA) pathway. Down-regulation of FPS also triggered a Fe-deficiency transcriptional response that is consistent with the Fe-deficient status observed in FPS silenced plants. Overall design: To induce FPS gene silencing, seedlings were germinated and grown for three days on sterile filter papers that were first placed on MS plates and subsequently transferred onto new MS plates supplemented (treatment) or not (control) with 30 µM MFZ for five days more. Three independent pools of seedlings (100 mg of FW) were collected per each treatment. Seedlings were ground to a fine powder using TissueLyser equipment and used for extraction of RNA. The quality and quantity of total RNA samples were assessed using a Bionalyzer Expert 2100 Instrument (Agilent Technologies) and an equimolar mixture of RNA samples from each treatment was prepared. The RNA samples (3 μg) were fragmented and ligated with adaptors prior to cDNA synthesis and PCR amplification. The cDNA libraries were prepared according to the Illumina protocols and sequenced using an Illumina HiSeq2000 machine to perform 2x100 paired-end sequencing.