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Species: Chara australis<br />
Organ … Species: Chara australis<br />
Organ: Internodal cells<br />
Organ specification: Vacuole and cytoplasm (interior of the cell excluding the vacuole)<br />
Amount: A single vacuole (approximately 25–50 μL) and the cytoplasm of a single cell.<br />
Growth conditions: C. australis was cultured in a plastic bucket, containing tap water and leaf mold extract, under fluorescent lamps with a 14-h light/10-h dark cycle in an air-conditioned room (25°C) at Tsuruoka Metabolome Campus, RIKEN laboratory.<br />
Experimental conditions: For habituation of C. australis, samples were transferred to another plastic bucket containing artificial pond water (APW) consisting of 0.1 mM each of KCl, NaCl, CaCl2, and 1 mM of NaHCO3 under a 12-h light/12-h dark cycle in chambers maintained at 25°C for at least 1 week.<br />
Sampling: A sample consisted of a single cell. Five different cells were sampled at each time point. Therefore, 5 replications were performed for each sample.<br />
Different light conditions: Five internodal cells were isolated at 7 time points (0, 3, 6, 12, 15, 18, 24 h after lighting).<br />
Continuous light/dark conditions: Five internodal cells were isolated at 0, 24, and 48 h after changing light conditions to continuous light or dark.<br />
CO2 deficiency: One bucket was filled with APW (control and +CO2); a second bucket, with APW without NaHCO3 (-CO2). After 36 h, 5 internodal cells were isolated.<br />
Heat stress: One bucket was stored at 25°C; a second bucket, at 37°C. Five internodal cells were isolated at 0, 24, and 48 h after application of heat stress.<br />
Sampling date: 1–2 September 2009 (different light conditions), 2–4 September 2009 (continuous light/dark conditions), 22 June 2009 (CO2 deficiency), and 15–17 February 2010 (heat stress).<br />
Isolation of vacuole and cytoplasm: A single internodal cell was isolated from neighboring cells and exposed to air on a paraffin wax board. After loss of turgor pressure, both ends of the cell were cut. By inclining the board, the vacuolar solution was collected and used as the vacuolar fraction. After removing the vacuolar solution completely, the remaining biomaterial of the cell was used as the cytoplasmic fraction.
Metabolism quenching method: Samples were immersed in liquid nitrogen within 30 s of sampling. After quenching, samples were stored in liquid nitrogen and extracted within 30 min.<br /> n and extracted within 30 min.<br />
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