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Articles

Photosynthetic Characteristics of Photoautotrophically Grown Tobacco Callus Cells ¹

Department of Biochemistry, The Connecticut Agricultural Experiment Station, New Haven, Connecticut 06504

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 -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.