Role of microRNAs in carbon metabolism and stress responses in Arabidopsis thaliana
Arabidopsis mutants (ucp1, ucp2, ucp3, ucp12, ucp13, ucp23, ucp123, and PAP1-D) and control (Col-0) seeds were surface sterilized with 70 percentage ethanol for 10 minutes, followed by 50 percentage bleach for 5 minutes, and then washed 6-7 times with autoclaved water for the sucrose treatment experiment. Sterilized seeds were then plated on 1/2 MS medium plates (half-strength Murashige and Skoog standard medium) minus sucrose with two segments of sterilized filter paper on top of the media. For stratification, the plates were maintained at 4 degree C for 72 hours. Plates were transferred to a working bench after stratification and allowed to grow in room temperature under continuous light for three days (72 hours). Half of the 3-day-old Arabidopsis seedlings were transferred aseptically to 1/2 MS medium plates containing 200 mM sucrose and grown for another 72 hours, while the other half remained in the germination media (1/2 MS medium minus sucrose) as a control. After 72 hours, sucrose-treated and control seedlings were harvested and frozen in liquid nitrogen and were used for total RNA and small-RNA extraction with the Spectrum Plant Total RNA Kit (Sigma-Aldrich) and mirPremier microRNA Isolation Kit (Sigma-Aldrich), respectively. RNA and small-RNA sequencing libraries were prepared in two biological replicates using small-RNA (100 nanogram) and total-RNA (1 microgram) inputs according to the instructions provided by TruSeq sRNA Sample Preparation Kit and Illumina TruSeq Stranded Total RNA with RiboZero Plant RNA-seq kit.