Single nuclei transcriptomic and bulk RNA-seq of Populus tremula x alba stem under well-watering and severe drought conditions

We have employed snRNA-seq using Singleron technology to dissect the molecular mechanisms controlling secondary xylem plasticity, which allows poplar trees to cope with severe drought. We generated four snRNA-seq libraries using mature stem sections, two per treatment. In this study, we present new molecular drivers of wood plasticity under drought, which allow trees to generate secondary xylem with more and narrower vessels. This trait confers a better adaptation under this abiotic stress. We also performed bulk RNA-seq under the same conditions to evaluate the accuracy of snRNA-seq in our analyses. Overall design: For the severe drought treatments, in vitro-cultivated hybrid poplars of WT, were transferred to pots containing peat, and grown under LD and 22°C conditions for 6 weeks. Six weeks after potting, the plants were divided into two groups: control and severe drought treatment. Irrigation was carefully controlled during the two weeks of treatment. Control plants were well-watered, keeping the solid moisture around Field Capacity (FC) (0.46 m3/m3) during the treatment. The weight of each pot was calculated at the FC before treatments. Severe drought treatment was achieved by stopping irrigation until the soil reached 15 % of its FC. The wilting threshold was reached when soil moisture decreased to 0.10 m3/m3. Then, a small amount of water was added per pot daily to keep the 15 % of FC by weighing the pots daily during 14 days. Nuclei isolation was performed from the internodes 14 and 15 from one plant, for each library, and snRNA-seq libraries were generated following the Singleron protocol. Bulk RNA-seq was extracted from trees under control and severe drought conditions from the stem. 3 biological replicates were generated per treatment.