OVEREXPRESSION OF TEMPRANILLO-LIKE PROTEINS PROMOTES DORMANCY RELEASE IN POPLAR

We investigated the effect of Populus TEML1 and TEML2 on the dormancy transcriptome through RNAseq experiments. We suggest these two genes play a role during poplar annual cycle, most specifically during dormancy based on their expression analysis. For this reason, we collected apical buds and generated bulk RNAseq libraries of overexpressing plants and their controls. We evaluated pathways which were altered in the transgenic lines and their role during the annual cycle. In vitro-cultivated hybrid poplars of TEML1ox, TEML2ox and WT were transfered to pots containing peat, and grown under LD and 22ºC conditions for 6 weeks. After this time, plants were moved to a short-day chamber for 8 weeks, where they ceased growth and set buds. After the 8 weeks of treatment, we collected the apical buds, grouping 3 buds per replicate, and 2 biological replicates. The remaining plants were subjected to a two-weeks of cold treatment and after this time, apical buds were collected following the short-day experimental design. RNA extraction was performed by adding to grinded frozen buds tissue CTAB extraction buffer with 2% β-mercaptoethanol, following by 3 washes with chloroform. After that, 7.5 M LiCl2:50mM EDTA was added, and samples were precipitated overnight at 4ºC. Next day, RNA was then purified according to the manufacturer’s instructions of NZY RNA purification kit (NZYTech, Lisboa, Portugal). A total of 100 ng of extracted RNA was used to generate the RNA-seq libraries. Libraries were generated from cDNA samples using the TruSeq Stranded mRNA Library Prep Kit (Nat Rev Genet. 2011 Sep 7;12(10):671-82) and they were sequenced on the Illumina platform (MACROGEN, Seúl, South Korea).