Methods
Plant material and establishment of photoautotrophic culture
Photomixotrophic callus cultures of Arabidopsis thaliana (ecotype Columbia [Col-0]) were grown in AT medium (B5 medium with 0.2 µM 2,4-D, pH 5.7) containing 2% sugarand 0.3% gelrite under 180 µEm-2s-1 of fluorescent light at 25°C. A photomixotrophic suspension culture was established from the green calli and cultivation was performed the presence of 2% sugaron a rotary shaker set to 120 rpm under the continuous fluorescent light (approximately180 µmol photons m-2s-1) at 25°C. The mixotrophic suspension culture cells were adopted to a elevated level of approximately 2% CO2 in air gas in special two-tiered flasks (Meyer and Spiteller, 1997; Sinha and Roitsch, 2002). The lower part of two-tiered flasks was filled with 50 mL carbonate buffer (2M K2CO3 and 2M KHCO3) which supported approximately 2% CO2 and 60 ml of cell suspension was cultivated in the upper part. The green cells were continuously sub-cultured to medium which contained lower content of sugar than before. Typically the sucrose concentration was reduced by 50%. After transfer to a lower sucrose concentration growth rates initially dropped. When the growth rate had recovered, typically after a period of several months, the next shift to a lower sucrose concentration was attempted. Finally, after a total period of about two years stable growth in a sucrose-free medium was achieved.For comparative analysis Arabidopsis seedlings were grown on soil for 10 days at RT and approx. 100 µmol photons m-2 s-1.
Heterotrophic cell suspension cultures
Arabidopsis (Arabidopsis thaliana), (ecotype Columbia [Col-0]), heterotrophic suspension cultures were maintained in Murashige and Skoog basal medium (Duchefa) containing 0.5 mg L21 naphthaleneacetic acid, 0.05 mg L21 kinetin, and 3% (w/v) glucose, pH 5.6. The cell suspension cultures were maintained in 250 mL conical flasks in the dark at 22°C in an orbital shaker. Subculture of 10 mL of culture to 100 mL of fresh media was conducted every 7 days. At day 4, cells in the middle of the log phase were collected and washed three times with water and frozen in liquid nitrogen.
Protein analysis
Total protein was extracted from frozen cell culture aliquots by grinding samples in protein extraction buffer (2 % (w/v) SDS, 56 mM NaCO 3, 12 % (w/v) sucrose, 56 mM DTT and 2 mM EDTA, pH 8.0). After heating at 70°C for 20 min and centrifugation at 16,000 g for 10 min total protein amount was determined using BCA protein assay reagent (Perbio Science) after trichloracetic acid precipitation. Proteins were separated on 12% polyacrylamide gels, blotted to Hybond-C Membranes (GE Healthcare) and probed with the specific antibodies using standard protocols (Sambrook and Russel, 2001). For comparison of proteins of Arabidopsis thaliana Col-0 (WT) and PA cell culture protein extracts were adjusted to equal amounts of protein.
Determination of porphyrins and pigments
Samples for chlorophyll and carotenoid measurement were ground in 80% acetone (v/v) with 10 µM KOH. Pigments were determined spectrophotometically as described before (Lichtenthaler 1987). Proto and Mg-porphyrins were extracted and analysed by HPLC following the method of Moulin et al. (2008) using a reverse phase column (Waters RP-18 ODS Hypersil 3 μM, 12.5 × 4 cm).
Extraction, derivatization and analysis of polar metabolites using GC-MS
Metabolite analysis by GC-MS was carried out exactly as described by Lisec et al., (2006).
Chlorophyll fluorescence measurements
Modulated chlorophyll fluorescence of the Arabidopsis cell suspension culture were measured using a PAM 2100 chlorophyll fluorometer (Heinz Walz, Effeltrich, Germany). Maximum PSII quantum yield of a dark-adapted sample (Fv/Fm) and effective PSII quantum yield of illuminated sample (ΦII;) were measured on cells dark adapted for 15 min as described by Schreiber et al., (1986). The preprogrammed protocol (Standard Run 3) was used for the determination of Fv/Fm and ΦII in a special cuvette designed for the purpose. The steady-state values obtained at the end of Run 3 were reported as the values of ΦII.