Matriz general de riesgos

2.2.2.1 Seed sterilization

Tobacco seeds were sterilized by 1 min incubation in 70% (v/v) ethanol followed by 5 min incubation in 5% (w/v) Dimanine C (Bayer, Leverkusen) solution. The sterilized seeds were washed 3 x in sterile water.

2.2.2.2 Plastid transformation

The biolistic transformation technique (Boynton et al. 1988, Svab et al. 1990, Svab and Maliga 1993) was applied. 60 mg of gold particles (0.6 µm; BioRad, USA) were suspended in 220 µl sterile water and mixed with 25 µl of purified plasmid DNA (1 µg/µl), 250 µl of 2.5 M CaCl2, 50 µl spermidin (0.1 M), followed by 2x washing of DNA-gold particles in 100%

ethanol (p.a.), and final suspension of DNA-gold particles in 72 µl of 100% ethanol. The gold particles were delivered into plant cells using the Particle Gun - PDS 1000/He, Bio-Rad (USA).

Parameters used with the Particle Gun

Helium pressure: 1100 psi

Rupture discs: 900 psi

Distance rupture disc/macrocarrier: 8 - 10 mm Distance macrocarrier/stopping screen: 10 mm Distance stopping plate/table: 7 cm

Vacuum: 0.85 bar

(26 – 27 inches Hg)

For each construct 20 leaves were shot and dissected 48 h after particle bombardment into 3 x 3 mm segments that were kept under non-selective conditions for 3 days, and then under selective conditions.

2.2.2.3 Selection and regeneration of mutants

Selection with 500 mg/liter of spectinomycin started three days after transformation. Green colonies began to appear and grow vigorously after approximately 3 weeks, while untransformed cell lines were bleaching and showed impaired growth. Resistant calluses were transferred to RMOP medium and cultivated in Petri dishes until shoot formation. For further segregation transplastomic lines were transferred into Petri dishes containing fresh medium at 3 - 4 week intervals. After several rounds of segregation, small green shoots of homoplastomic lines were transferred to 750 ml glass jars containing B5 agar with antibiotic, and grown for 4 - 6 weeks until use.

2.2.3 Isolation and fractionation of chloroplasts

2.2.3.1 Isolation of intact chloroplasts

Isolation of intact plastids was performed according to Müller and Eichacker (1999).

Homogenization Medium 0.4 M sorbitol

50 mM HEPES-KOH, pH 8.0 2 mM EDTA, pH 7.5

Percoll Gradient Solution 45% / 85% Percoll (w/v) 0.4 M sorbitol 50 mM HEPES-KOH, pH 8.0 2 mM EDTA, pH 7.5 Washing Buffer 0.4 M sorbitol 50 mM HEPES-KOH, pH 8.0

10 - 20 g of freshly harvested leaf material was homogenized in approximately 100 ml of homogenization medium. The homogenate was filtrated through two layers of Miracloth (100 µm, Calbiochem, La Jolla, USA), and centrifuged at 4000 x g for 1 min. The sediment was carefully suspended in 1 ml of homogenization medium and loaded onto 45 - 85% Percoll gradients. The gradients were prepared by loading 10 ml of 45% and 5 ml of 85% Percoll in Corex tubes. After centrifugation of 8 min at 4000 x g in a “swing out” rotor the lower band containing intact plastids was collected using a Pasteur pipette and washed once in washing buffer (centrifugation 3 min, 1000 x g). All preparation steps were performed at 4°C, and isolated plastids were stored in darkness on ice.

2.2.3.2 Fractionation of chloroplasts into stroma and thylakoid membranes

TMK Buffer

10 mM Tris-HCl, pH 6.8

10 mM MgCl2

20 mM KCl

Freshly prepared chloroplasts were osmotically ruptured with TMK buffer in a ratio of 100 µg chlorophyll to 0.5 ml buffer. The chloroplasts were lysed for 10 min in darkness on ice and centrifuged for 3 - 5 min at 2000 x g. The supernatant (stroma fraction) was collected, the pellet (thylakoids) was washed twice by centrifugation in TMK buffer. The final pellet was resuspended in TMK buffer. Aliquots of the thylakoids were stored at –70°C.

2.2.3.3 Isolation of the major thylakoid protein complexes

The major thylakoid protein complexes, equivalent to 3 x 108 chloroplasts were solubilized 10 min on ice by 1.5% (w/v) β-dodecylmaltoside (final concentration) as described by Müller and Eichacker (1999). The thylakoid lysate was loaded onto linear 0.1-1.0 M sucrose gradients and centrifuged for 16.5 h at 36 000 x g at 4°C (Beckman “swing out” rotor SP 40 Ti). The sucrose gradients were top-to-bottom fractionated into 35 fractions of 300 µl each using an ISCO 640 gradient fractionator (Instrumentation Specialties Company, USA). For further analyses individual gradient fractions were precipitated with 10% (w/v) trichloroacetic acid and washed with 100% acetone.

2.2.3.4 Washing of thylakoid membranes with Na2CO3 HS Buffer 0.1 M sucrose 10 mM HEPES-KOH, pH 8.0 Carbonate Buffer 0.1 M Na2CO3 0.1 M sucrose 10 mM HEPES-KOH, pH 8.0

To dissociate peripherally associated proteins from thylakoid membranes a Na2CO3 washing

step was applied. Thylakoid samples at a concentration of 0.5 mg chlorophyll/ml were washed with HS buffer and incubated with 100 µl Na2CO3 for 10 min on ice. Washed

thylakoids were pelleted by centrifugation for 30 min at 18 000 x g and suspended in an appropriate volume of HS buffer.

2.2.3.5 Purification and fractionation of chloroplast envelope membranes

Tricine Solution (1l) 20 ml 0.5 M Tricine, pH 7.9 2 ml 0.5 M EDTA, pH 8.0 72 µl β-mercaptoethanol 1 ml PMSF 0.65 M sucrose (200 ml) 44.48 g sucrose 4.0 ml 0.5 M Tricine, pH 7.9 14.4 µl β-mercaptoethanol 0.2 ml PMSF

Sucrose Gradients (each 200 ml) 0.996 M 0.800 M 0.465 M sucrose 68.57 g 54.76 g 31.82 g 0.5 M NaiPO4, pH 7.9 4 ml 4 ml 4 ml 0.5 M EDTA, pH 8.0 0.4 ml 0.4 ml 0.4 ml NaiPO4 Buffer Stock 93.2 ml 0.5 M Na2HPO4 6.5 ml 0.5 M NaH2PO4 pH 7.9 NaiPO4 Buffer (1l) 20 ml 0.5 M NaiPO4 Buffer Stock, pH 7.9 72 µl β-mercaptoethanol 1 ml PMSF

Purified intact plastids (see Section 2.2.3.1) were suspended in 10 ml of 0.65 M sucrose and ruptured by homogenization in a Dounce homogenizor on ice. The suspension of broken chloroplasts was filled up to 40 ml with Tricine solution and centrifuged for 1 h at 186 000 x g (Beckman TT45 rotor) at 4°C. The thylakoid pellet containing the envelope fraction was then suspended in Tricine solution and loaded onto sucrose gradients consisting of 7 ml 0.996 M / 10 ml 0.800 M / 8 ml 0.465 M sucrose solutions. Envelope membranes were isolated by centrifugation at 113 000 x g (Beckman SW27 rotor) for 3 h at 4°C. During this centrifugation step envelope membranes separate into three bands: outer envelope + RuBisCo (in 0.465 M sucrose), outer envelope (0.465 M/0.800 M sucrose), inner envelope (0.800 M/0.966 M sucrose), and thylakoid membrane pellet. Inner and outer envelope membranes were collected and washed 1:3 in NaiPO4 buffer by 1 h centrifugation at 113 000 x g at 4°C.