Type 11 optical unit

Prior to lysis, cells were treated with 100 µg/ml cycloheximide (except for splitting assays, see below) for 5 minutes. Cells were washed in ice-cold PBS (supplemented with 100 µg/ml cycloheximide), lysed in polysome lysis buffer B (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 1 mM DTT, 40 U/ml RNasin Plus Rnase Inhibitor, Dnase I, Pierce Protease and Phosphatase inhibitors, 100 µg/ml cycloheximide), and crude lysates were centrifuged for 10 min at 13,000 x g to pellet debris. Clarified lysates were layered on 10–50% sucrose gradients (prepared in 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl₂) and centrifuged for 2 h at 190,000 × g. Gradients were fractionated using an NE-1000 syringe pump (New Era Pump Systems, Inc.) and 254 nm absorbance was measured using an ISCO Type 11 optical unit and UA-6 detector.

All buffers were described in [39 (link)] and all steps performed at 4°C. Briefly, 1 to 2 g of frozen powder from non-treated (22°C), heat treated (37°C for 5 h and 24 h) and recovered (R22°C for 5 h and 24 h) seedlings were suspended in 4 mL of PEB, incubated on ice for 30 min and centrifuged 15 min at 16,000 g to remove debris. Samples were then filtered on 0.2 µM filters (Sarsted), loaded on 8 mL sucrose cushions, and centrifuged in a Beckman SW41 rotor for 18 h at 35,000 rpm. Pellets were re-suspended in 1 mL of RB and kept on ice for 30 min. A short centrifugation (2 min at 5,000 g) was performed to remove the last debris. Finally, 1 mL of supernatant was layered onto 9 mL linear 15–60% sucrose gradients and centrifuged at 38,000 rpm for 6h30. Gradients were fractionated using the Type 11 Optical Unit (Teledyne ISCO) system and a UA-6 UV/VIS Detector (Teledyne ISCO) at 254 nm. Values and ratios for 40S and 60S/80S peaks are available in Table S4.