Cryolys

The proteome of four biological replicates of mature and immature stem/leaf phytomers were analyzed. Proteins were extracted by lysing 100 mg of chopped mature or immature tissues with 2% SDS, 6.4 M urea, 0.1 M Tris-HCl, and 0.1 M dithiothreitol buffer at pH 8.5. Therefore zirconia beads (0.5 mm diameter) were added to the samples and homogenized (Precellys 24, Bertin) using a liquid nitrogen cooler (Cryolys, Bertin). The homogeniser operated at 11°C with 3 cycles of 45 s at 6500 rpm, with 30 s intervals between cycles. Samples were then centrifuged at 13,000 rpm for 10 min at 4°C, and the protein concentration in the supernatant was measured using the 2D Quant Kit (GE Healthcare).

Cell pellets derived from 500 ml exponentially growing cultures of yeast strains expressing TAP-tagged proteins were resuspended in 10 ml Buffer P1 (150 mM KAc, 20 mM Tris, pH 8.0, 5 mM MgCl₂, 1 mM DTT, 0.2% (w/v) Triton) supplemented with Protease Inhibitors and RNasin (Promega). Mechanical cell lysis was performed at 4°C for 6 × 30 s at 6000 rpm with 5 × 30 seconds pausing in a Precellys Evolution coupled to Cryolys (Bertin Instruments). Cell debris were pelleted by centrifugation at 18 000 g for 15 min at 4°C and clarified lysates were used for western blot analysis and for affinity purification.

Samples of bacterial cultures in biological triplicates were harvested at given time points^{13 (link),62 (link)}. For intracellular metabolites, an equivalent of 10 mL culture at an OD₆₀₀ of 0.2 was rapidly filtered on a 0.22 µm nitrocellulose filter (Millipore). The filter papers were immediately placed in 2-mL screw cap tubes of 1 mL extraction solvent containing 20%/40%/40% (vol/vol) water/acetonitrile/methanol (Fisher) with approximately 500 µL of silica beads at −20 °C. The samples were homogenized twice in a Precellys Evolution (Bertin) at 4,500 rpm for 45 seconds, cooled to 5 °C using a Cryolys (Bertin), and with 5 min rest between cycles. Samples were centrifuged, and 750 µL of the extract was filtered through 0.22-µm Spin-X centrifuge filters (Corning, Life Technologies) at 10,621 g for 3 min, and stored at −80 °C. For extracellular metabolites, 1 mL of culture was collected and centrifuged, and 750 µL of supernatant was filtered twice through 0.22-µm Spin-X columns. Detergent in the samples were removed using Pierce detergent removal spin columns (Thermo), according to manufacturer’s instructions. Metabolites were extracted by adding 200 µL sample to 800 µL of 50%/50% (vol/vol) acetonitrile/methanol (Fisher), the samples were spun down, as before, and the supernatant collected and stored at −80 °C until analysis.