Photosynthetic Characteristics of Photoautotrophically Grown Tobacco Callus Cells

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Abstract
Haploid callus cells of tobacco (Nicotiana tabacum) were grown photoautotrophically on a solid agar medium in the absence of sucrose in Petri plates in an atmosphere of 1% or 3% CO2 in air. The averages of dry weight increases for four to five consecutive passages were 2.3- to 3.6-fold per 3-week passage for different subclones. Photosynthetic 14CO2 assimilation was maximum at about 1% CO2 with half-maximal rates obtained at 0.2% CO2. At saturating CO2 concentration the average rate of CO2 fixation was about 5 μmole per gram fresh weight per hour or about 125 μmole per mg of chlorophyll per hour. The existence of an active photorespiratory system in these tissues was established in a number of independent ways. The photosynthetic rate in 0.18% CO2 was inhibited 38 to 50% in 100% O2 compared with 21% O2. Glycolate accumulated at a constant rate in the presence of 5 mm α-hydroxy-2-pyridinemethanesulfonic acid for 20 minutes in light. This rate was rapid relative to the photosynthetic rate. Glycolate synthesis was three times faster in autotrophic than in heterotrophic cells. [1-14C]Glycolate was rapidly metabolized and the products included 14CO2, [14C]glycine, and [14C]serine, thus demonstrating an active glycolate pathway. Photorespiration was demonstrated directly by measurement of an O2-dependent release of 14CO2 in the light from callus that fixed 14CO2 for about 22 hours. Autotrophic growth in 60% O2 and 0.03% CO2 was slowed and ceased entirely after two or three passages, while heterotrophic growth was unaffected by 60% O2 in the atmosphere. The method of growing autotrophic callus which has an active photorespiratory system should facilitate the selection and analysis of photosynthetic mutants in which photorespiration is regulated.