Effect of shade and nitrogen content on Arabidopsis Col-0 and cytokinin mutants abcg14 and cypDM
Effect of shade and nitrogen content on Arabidopsis Col-0 and cytokinin mutants abcg14 and cypDM
For each sample, around 20 whole shoots of 4-day old Arabidopsis thaliana seedlings, ecotype Col-0, were grown in longday conditions (16 h light : 8 h dark, PAR=150, 70% humidity, R:FR=2.5 at 20°C) harvested at Zeitgeber Time (ZT) 8, and directly flash frozen in liquid nitrogen. 12 unique samples were collected, with three biological repetitions each, for a total of 36 samples. The variations for each sample are as follows. Each possible combination has been harvested. Genotype: Wild-Type (Col-0), abcg14 (Ko et al., 2014), cypDM (cyp735a1,a2, Kiba et al., 2013). Light quality: White Light (WL, R:FR=2.5), White Light + Far Red light (WL+FR, R:FR=0.25). Nitrate availability: High Nitrate (HN, 10mM KNO3), Low Nitrate (LN, 0.2mM KNO3). Plants were grown for 4 days under WL conditions and on the different nitrate conditions. Plants from the WL+FR samples were treated for 90min prior the collection with WL+FR. Arabidopsis thaliana seeds were sterilized and sowed in vitro on a modified 1/2MS medium where the NH4NO3 nitrogen source was replaced with either 10mM KNO3 (HN) or 0.2mM KNO3 + 9.8mM KCL (LN). The other nutrients composition and concentration are as follow: CaCL2 1.5mM, MgSO4 0.75mM, KH2PO4 0.625mM, Fe(III)Na-EDTA 50µM, MnSO4 50µM, H3BO3 50µM, ZnSO4 15µM, Na2Mo4 0.5µM and CuSO4 0.1 µM. Medium was supplemented with 1g/L 2-(N-morpholino)ethanesulfonic acid (MES), 1% Plant Agar (Duchefa Biochemie) and pH adjusted to 5.8 with KOH. After a 3 days stratification period (4°C, in the dark), seeds were transferred in a growth chamber with controlled conditions: long days photoperiod (16h light, 8h dark), PAR=150, 70% humidity, 20°C, R:FR=2.5 for WL conditions and R:FR=0.25 for WL+FR conditions.  Plates were placed in a modified D-root system (Silva-Navas et al., 2015), where roots are shielded and only shoots are exposed to the light.   Samples were grounded to powder with a bead basher and RNAs>200nt were extracted using a Qiagen RNAeasy Minikit, following manufacturer's instructions. Residual DNA was removed using the on column Qiagen RNAse-free DNAse, following manufacturer's instructions. Total RNA was sent to Novogene for QC, library construction and sequencing. Briefly, messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers. Then the second strand cDNA was synthesized using dUTP, instead of dTTP. The directional library was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection.