Approaches to evaluating the extent to which guard cell protoplasts of Nicotiana glauca (tree tobacco) retain their characteristics when cultured under conditions that affect their survival, growth, and differentiation

Taylor, J. E.; Abram, B.; Boorse, G.; Tallman, G.

doi:10.1093/jxb/49.special_issue.377

Guard cell protoplasts (GCP) of Nicotiana glauca (Graham), tree tobacco, were cultured at 32 degrees or at 38 degrees C in media containing or lacking 0.1 micromolar ABA. Cells cultured at 32 degrees C exhibited large increases in cell volume, dedifferentiated, and divided (Type II cells). Cells cultured at 38 degrees C increased less in volume, retained the general morphology of guard cells, and did not divide (Type III cells). Cells cultured at 38 degrees C in media containing ABA (Type IV cells) neither grew nor divided; they retained the morphology of freshly-isolated GCP (FGCP). Experiments were performed to determine the extent to which selected cell types retained certain physiological, biochemical, and molecular characteristics of FGCP. Type IV cells increased in volume by 40% when illuminated with low-intensity blue light (15 micromole m-2 s-1) over background red light (300 micromole m2 s-1). Blue light-induced swelling of GCP was inhibited fully by 10 MM m-chlorophenylhydrazone and 5 mM dithiothreitol. Chloroplasts of Type II cells underwent senescence. The capacity of chloroplasts of Type III and Type IV cells for photochemical quenching of Chl a fluorescence was reduced compared to that of FGCP, and cultured cells lost all capacity for non-photochemical quenching. Zeaxanthin (Z) has been identified as the putative blue light photoreceptor of guard cells. Type III cells lost capacity for light-induced Z formation, but Type IV Cells retained the capacity to form Z. Results of differential display-PCR indicated that the greatest number of absolute differences in PCR products was between FGCP and Type II cells; the fewest number was between FGCP and Type IV cells. Both the blue light photoreceptor and the signal transduction pathway linking blue light photoreception to activation of the plasma membrane H+-translocating ATPase of guard cells remain intact in cultured Type IV cells. Culture conditions alter both photochemical and nonphotochemical processes of chloroplasts of Type III and Type IV cells, but chloroplasts of Type IV cells retain the capacity to acidify the thylakoid lumen to activate a functional violaxanthin-antheraxanthin de-epoxidase in the thylakoid membrane. The data support the hypothesis that each cell type exists in a differentiated state more or less similar to that of FGCP, with Type II cells having the least similarity, Type III cells having greater similarity, and Type IV cells having the greatest similarity.