On the use of Avena protoplasts to study chloroplast development

Different methods were tested to isolate protoplasts from etiolated, partially greened, and light-grown leaves of Avena sativa. Preparations with high yields and high photosynthetic capacities (time of illumination ≧4 h) were obtained when small transverse leaf segments were incubated for 2 h at 30°C in 2% cellulysin (Calbiochem), 0.6 M mannitol, and 0.5% bovine serum albumin (BSA) at pH 5.6, without shaking. As measured by light-dependent O2 evolution or fixation of labeled bicarbonate, protoplasts exhibited rates of up to 124 μmol per mg of chlorophyll per h at 20°C and saturating bicarbonate, which were nearly identical to those found with intact leaves. The assay conditions necessary for this activity were 0.6 M sorbitol, 50 mM N-2-hydroxy-ethylpiperazine-N'-2-ethane sulfonic acid (pH 7.6), and 10 mM NaHCO3. If plastids were isolated from these protoplasts, sorbitol was 0.45 M, including 10 mM ethylenediaminetetraacetate (EDTA). under these conditions, rates of photosynthesis were up to 125 (light-grown) and 71 (6 h illuminated) μmol O2 evolved or (14)CO2 fixed per mg of chlorophyll per h, compared to 3.5 μmol·mg chl(-1)·h(-1) obtained with mechanically isolated plastids. With this system, CO2-dependent O2 evolution was already detected after 3 h of illumination of etiolated tissue, but could only be observed at pH values between 7.6 and 8.6, in the presence of EDTA. At lower pH (7.3) or at pH 7.6 in the absence of EDTA, light-dependent O2 evolution up to 24 h of greening was only measurable with 3-phosphoglycerate as the substrate. The possible effects of EDTA in this respect as well as the advantages of using protoplasts or plastids isolated from protoplasts for developmental studies are discussed.