Influence of carbon source and CO 2-enrichment on biochemical parameters associated with photomixotrophia in maize callus cultures

Abstract

Summary To learn about the biochemical processes underlying the induction of photomixotrophia in maize cell culture, maize calli were cultured in medium containing either glucose or starch as the carbon source. The effect of a CO 2 -enriched atmosphere on different parameters was tested. Levels of chlorophyll and CO 2 -fixing enzymes were measured to assess the greening process concomitant to histological observations of chloroplast development. Both starch and glucose promoted higher chlorophyll accumulation in callus cultured under light than sucrose. Histological analysis of green callus grown on glucose-containing medium revealed the formation of poorly developed chloroplasts containing starch grains, whereas in starch medium a large number of elongated chloroplasts containing thylakoids were observed. Exposure of these calli to a CO 2 -enriched atmosphere enhanced the plastid differentiation process up to mature chloroplasts with grana and intergranal thylakoids. Western-blot analysis demonstrated the presence of CO 2 -fixing enzymes, Rubisco (EC 4.1.1.39) and PEP carboxylase (EC 4.1.1.31), as well as Rubisco activase in greening callus. Rubisco and PEP carboxylase activities showed large values when starch was the carbon source in the medium. Results of histological analysis and a/b chlorophyll ratios indicated that the chloroplasts formed were of the C 3 -type. PEP carboxylase kinetic properties were also consistent with a C 3 -type enzyme involved in anaplerotic functions. It is concluded that under the experimental conditions tested, starch plus CO 2 -enriched atmosphere are the best carbon source for inducing and supporting photomixotrophia in maize cultures, as indicated by several biochemical parameters.