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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

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<mark>Journal publication date</mark>15/04/1998
<mark>Journal</mark>Journal of Experimental Botany
Issue numberSPEC. ISS.
Number of pages10
Pages (from-to)377-386
Publication StatusPublished
Early online date1/03/98
<mark>Original language</mark>English


Guard cell protoplasts (GCP) of Nicotiana glauca (Graham), tree tobacco, were cultured at 32 °C or at 38 °C in media containing or lacking 0.1 μM ABA. Cells cultured at 32 °C exhibited large increases in cell volume, dedifferentiated, and divided (Type II cells). Cell cultured at 38 °C increased less in volume, retained the general morphology of guard cells, and did not divide (Type III cells). Cells cultured at 38 °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 μmol m-2 s-1) over background red light (300 μmol m-2 s-1). Blue light-induced swelling of GCP was inhibited fully by 10 μM 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.