Photosynthetic Characteristics of Photoautotrophically Grown Tobacco Callus Cells

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% CO(2) 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 (14)CO(2) assimilation was maximum at about 1% CO(2) with half-maximal rates obtained at 0.2% CO(2). At saturating CO(2) concentration the average rate of CO(2) fixation was about 5 mumole per gram fresh weight per hour or about 125 mumole 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% CO(2) was inhibited 38 to 50% in 100% O(2) compared with 21% O(2). Glycolate accumulated at a constant rate in the presence of 5 mm alpha-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-(14)C]Glycolate was rapidly metabolized and the products included (14)CO(2), [(14)C]glycine, and [(14)C]serine, thus demonstrating an active glycolate pathway. Photorespiration was demonstrated directly by measurement of an O(2)-dependent release of (14)CO(2) in the light from callus that fixed (14)CO(2) for about 22 hours. Autotrophic growth in 60% O(2) and 0.03% CO(2) was slowed and ceased entirely after two or three passages, while heterotrophic growth was unaffected by 60% O(2) 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.