BioSample

Protocols: Xylem, inner bark (including phloem and cortex), and outer bark (phellem) layers were manually peeled from the first 10 cm segments of the main stem taken above root collar and frozen in liquid nitrogen. Phellem and inner bark layers were collected using the natural breaking regions occurring at the vascular cambium and phellogen, by quickly stripping the full bark layer. Debarked stems were immediately placed in liquid nitrogen and developing xylem was collected by scraping the wood with a scalpel blade. Plants were grown in a glasshouse, in 7 L pots containing a mixture of soil and sand (1:1); well watered plants were treated with 300ml water/day for 18 months; water deficit plants were treated with 300ml water/day for 12 months followed by water witholding for 1 month and 5 months with 300ml water/week. Frozen samples were ground with a mortar and pestle in liquid nitrogen and RNA extraction was performed according to (Reid et al., 2006; DOI:10.1186/1471-2229-6-27) with minor modifications described in (Leal et al., 2022; DOI:10.1093/treephys/tpab176) 1)mRNA enrichment and purification: Oligo dT Selection to enrich the mRNA; 2)RNA fragment and reverse transcription; 3)End repair, add A and adaptor ligation; 4)PCR;5)Single strand separation and cyclization; 6)DNA nanoball synthesis