Multifaceted Activity of Cytokinin at Distinct Phases of Leaf Development Shapes Its Size and Structure in Arabidopsis thaliana

We performed a transcriptome profiling on the leaf 3 microdissected from plants with altered cytokinin levels 3h after activation at 9 DAS, when this leaf is normally fully composed of proliferating cells to get an insight into dynamics of molecular network underlying cell responses to CK excess and deficiency in proliferating cells. Seeds of Arabidopsis thaliana transgenic lines (CaMV35S>GR>ipt and CaMV35S>GR>HvCKX2) and corresponding wild-types (Col-0) were sown on nylon meshes placed on half-strength Murashige and Skoog (MS) medium containing 0.5% (w/v) MES and 0.8% (w/v) agar. After 2 days of stratification, plants were removed to growing chambers with 16-h day/8-h night, 21 C and 19 C, respectively, under continuous light (110 mol m-2 s-1). DEX-inducible lines and wild-types were activated at 9 DAS by transfer of nylon meshes to half-strength MS medium supplemented with either 2.5 M DEX dissolved in 5x10-4% (v/v) DMSO or just 5x10-4% (v/v) DMSO (mock). The leaf 3 was dissected from i > 250 individual seedlings per variant on a cooling plate under a stereomicroscope with precision needle and removed into the liquid nitrogen. For RNA-seq 2 biological replicates of WT, ipt and HvCKX2 activated by DEX were used. The RNA was extracted according the same method described for qRT-PCR. RNA sequencing was done by GATC Biotech AG (Konstanz, Germany).