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

Guard cell protoplasts (GCP) of Nicotiana glauca (Graham), tree tobacc o, were cultured at 32 degrees C or at 38 degrees C in media containin g or lacking 0.1 mu M ABA. Cells cultured at 32 degrees C exhibited la rge increases in cell volume, dedifferentiated, and divided (Type II c ells). Cells cultured at 38 degrees C increased less in volume, retain ed 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 fr eshly-isolated GCP (FGCP). Experiments were performed to determine the extent to which selected cell types retained certain physiological, m olecular characteristics of FGCP. increased in volume by similar to 40 % when illuminated with low-intensity blue light (15 mu mol m(-2) s(-1 )) over background red light (300 mu mol m(-2) s(-1)). Blue light-indu ced swelling of GCP was inhibited fully by 10 mu M m-chlorophenylhydra zone 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 Chi a fluorescence was reduced compare d to that of FGCP, and cultured cells lost all capacity for non-photoc hemical 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 gre atest number of absolute differences in PCR products was between FGCP and Type II cells; the fewest number was between FGCP and Type IV cell s. Both the blue light photoreceptor and the signal transduction pathw ay linking blue light photoreception to activation of the plasma membr ane H+-translocating ATPase of guard cells remain intact in cultured T ype IV cells. Culture conditions alter both photochemical and nonphoto chemical processes of chloroplasts of Type III and Type IV cells, but chloroplasts of Type IV cells retain the capacity to acidify the thyla koid lumen to activate a functional violaxanthin-antheraxanthin de-epo xidase 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 grea test similarity.