12003:

Phosphoinositide turnover is involved in hormone-induced tracheary element differentiation in Zinnia elegans L. cells.

Authors:	Zhang, Xianchao(A)Coté, Gary, G.(B)Crain, Richard, C.(A)
Affiliations:	(A): Molecular & Cell Biology, University of Connecticut (B): Biology, Radford University
Presenter:	Zhang, Xianchao , XIZ94001@uconnvm.uconn.edu

Mesophyll cells of Zinnia elegans L. cv. Envy, when cultured in the presence of auxin and cytokinin, differentiate into tracheary elements (TEs). Differentiation involves production of secondary cell wall and programmed cell death as mature TEs are hollow conducting vessels of the xylem. Ca2+ may play a role in this differentiation since Ca2+ accumulates in ER-like structures during differentiation, and since Ca2+ channel blockers, calmodulin antagonists, and reduced extracellular Ca2+ levels all inhibit TE formation. We found that extracellular Ca2+ is required early in TE differentiation. If 300 mM Ca2+ was added to the medium at different times after establishing the culture, the maximum number of TEs could be obtained when Ca2+ was added within the first 6 h of culturing. Levels of Ca2+ less than 1 mM could not trigger TE differentiation. Levels greater than 300 mM did trigger differentiation, but the process was delayed. If cultures were initiated in 300 mM extracellular Ca2+ and then transferred to 0.5 mM extracellular Ca2+, TE differentiation was inhibited, and the earlier the transfer the fewer the TEs which could be recovered, particularly in the first 6 h of culturing. If cells were cultured in plasticware to reduce leaching of Ca2+ into the medium no TEs developed when cells were transferred from 300 mM Ca2+ to Ca2+-free medium, even if the transfer was after 48 h, suggesting that a minimal level of extracellular Ca2+ is required throughout the differentiation process. We found an increase in phospholipase C (PLC) activity and inositol 1,4,5-triphosphate (IP3) levels 4 h after application of auxin and cytokinin to the culture. The PLC inhibitors U73122 and U73343 prevented the increase in IP3 levels and inhibited TE differentiation, with similar dose-response curves. Furthermore, pertussis toxin, a G-protein inhibitor, both blocked IP3 accumulation and inhibited TE differentiation. These data suggest a model in which auxin and/or cytokinin triggers TE differentiation by activating a G protein that stimulates PLC activity, leading to production of IP3, and subsequent mobilization of Ca2+. Preliminary data from experiments using cGMP agonists or LY83583, a guanylyl cyclase inhibitor, suggest that cGMP production may also be involved in signal transduction leading to TE differentiation. Supported by the NSF, USDA, and the US-Israeli Binational Agricultural Research and Development Fund.