Poster: Cell Cycle

Abs # 236: Elevated Temperature Inhibits the G1-to-S Phase Cell Cycle Transition in Cultured Guard Cell Protoplasts (GCP) of Nicotiana glauca (Graham).

Presenter:	Tallman, Gary , gtallman@willamette.edu
Additional Authors	Hayashi, Derek  (A)   Gushwa, Nathan  (A)   Kemper, Andrea  (A)   Pullen, Sam  (A)   Fiedler, Sarah  (A)   Miller, Linnsey  (A)   Upton, Jason  (A)   Tay, Chloe F (A)
Affiliations:	(A): Willamette University

When guard cell protoplasts (GCP) of Nicotiana glauca (Graham) are cultured at 32°C in media containing an auxin and a cytokinin, they re-enter the cell cycle, de-differentiate, and divide to form embryogenic callus from which plants can be regenerated. GCP of N. glauca cultured at 38°C grow, but they do not de-differentiate fully, nor do they divide. GCP of N. glauca cultured at 38°C in media containing 0.1 µM abscisic acid (ABA) remain differentiated in culture. To determine whether elevated temperature and/or elevated temperature and ABA prevent GCP from entering S phase of the cell cycle, GCP were evaluated for capacity to incorporate BrdU into replicating DNA at 32°C, at 38°C, or at 38°C in a medium containing 0.1 µM ABA. Initially and at the end of each successive 24 h period over two weeks, BrdU was added to a final concentration of 25 nM to multiple primary cultures established from a single GCP isolate. After another 24 h of culture, nuclei were isolated, fixed, and doubly-stained with FITC-conjugated rabbit anti-BrdU antibody and with Hoechst 33342. Nuclei were visualized initially with Hoechst and then scored for BrdU incorporation. When GCP were cultured at 32°C, BrdU incorporation into nuclear DNA was detected within 48-72 h. The percentage of nuclei with BrdU-labeled DNA was 18 ± 1.3% (mean; se; n = 3) after 72 h. Incorporation ranged from 24.9-30.1% over days 4-7 and then declined steadily to < 5% after 10 days. GCP cultured at 38°C in media lacking or containing 0.1 µM ABA survived in high percentages (70-80%) but did not incorporate BrdU. Elevating culture temperature from 32 to 38°C thwarts hormone-dependent re-entry of cultured GCP of N. glauca into the cell cycle by blocking the G1-to-S phase transition. Supported by NSF MCB-9900525.