Raised level of cyclin dependent kinase A after prolonged suspension culture of Nicotiana plumbaginifolia is associated with more rapid growth and division, diminished cytoskeleton and lost capacity for regeneration: implications for instability of cultured plant cells

Abstract

A long maintained suspension culture of Nicotiana plumbaginifolia, compared with a newer culture, had increased levels of cyclin dependent kinase A (CDKA;1) a halved cell doubling time and twice the stationary cell density. The newer culture downregulated CDKA protein levels by 75% during the transition to stationary phase and, in mixtures of natural and synthetic auxins with cytokinins, developed shoot tissue that could regenerate into plants, while the older cell line developed only callus. The older cell line retained some posttranslational regulation of CDK catalytic activity that was indicated by lower CDK enzyme activity in stationary phase and by a peak profile of CDK enzyme activity at mitosis in synchronous culture. Only the older cell line lacked preprophase band (PPB) microtubules and took 1 h less time after S phase to reach cytokinesis. It is suggested that the raised CDK activity after prolonged culture has multiple effects, including a marked shortening of G1 phase by a universal mechanism of CDK activation of transcription factors for DNA synthesis, and prevention of PPB assembly consistent with earlier observed effects of CDK microinjection, as well as a predicted elevation of mutagenesis by opposition of checkpoint arrest. Proliferation remained in balance with faster growth, which may have been accelerated by CDK activation of transcription factors. It is concluded that cells in which mutations elevate CDK expression and accelerate proliferation will inevitably predominate when populations are propagated by mass transfer. High CDK activity in such cells obstructs plant regeneration by preventing assembly of the PPB cytoskeleton and by countering restriction of cell division to meristems and the termination of proliferation that is required for organ development.