Two Immunological Approaches to the Detection of Ribulose-1,5-Bisphosphate Carboxylase in Guard Cell Chloroplasts

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Two immunological approaches were used to determine if ribulose bisphosphate carboxylase oxygenase (RuBisCo) is present in guard cell chloroplasts. Immunocytochemistry on thin plastic sections using tissue samples that were processed using traditional glutaraldehyde/osmium fixation and then restored to antigenicity with metaperiodate treatment, resulted in labeling over wild-type mesophyll and guard cell plastids of several green and white variegated Pelargonium chimeras. The density of immunogold labeling in guard cell chloroplasts was only about one-seventh of that noted in mesophyll chloroplasts on a square micron basis. Because guard cell chloroplasts are much smaller than mesophyll chloroplasts, and occur at lower quantities/cell, the relative differences in RuBisCo concentration between the cell types indicate that guard cells have only 0.48% of the RuBisCo of mesophyll cells. No reaction was noted over 70S ribosomeless plastids of these chimeras even though adjacent green chloroplasts were heavily stained, indicating the high specificity of the reaction for RuBisCo. Spurr's resin gave the most successful colloidal gold labeling in terms of low background staining and structural detail but L. R. White's resin appeared to be superior for antigen retention. In the white leaf edges of the white and green Pelargonium chimeras, the only green, functional chloroplasts are in the guard cells. When either whole tissue or plastid enriched extracts from this white tissue were electrophoresed, blotted, and probed with anti-RuBisCo a large subunit band was detected, identical to that in the green tissue. These data indicate that a low, but detectable, level of RuBisCo is present in guard cell chloroplasts.

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