Characterization of cell type specific gene expression in Nicotiana tabacum leaf tissue.

Abstract

Protoplasts isolated from Nicotiana tabacum leaf tissue were characterized through flow cytometric analysis based on chlorophyll autofluorescence and cell size. For mesophyll protoplasts, a high correlation coefficient (r² = 0.973) was observed between chlorophyll content and flow cytometric measurement of chlorophyll fluorescence. Chlorophyll fluorescence was found to correlate with chloroplast number (r² = 0.992) in protoplasts isolated from a single leaf, however comparison of these same parameters between experiments utilizing different leaves yielded a poor correlation. Cell size measured using pulse-width time-of-flight generated from the chlorophyll autofluorescence signal was highly correlated with cell diameter (r² > 0.99). Comparison of red fluorescence generated pulse-width time-of-flight size measurements with chloroplast number was also highly correlated with r² = 0.986. Fluorescein diacetate fluorochromasia generated pulse-width time-of-flight can be used to separate protoplasts which contain chloroplasts from protoplasts devoid of chloroplasts when analyzed with chlorophyll autofluorescence in a two-parameter histogram. Separation of the population devoid of chloroplasts on the basis of pulse-width time-of-flight provides an accurate means of measuring cell diameter. The purification of homogeneous populations of protooplasts derived from photosynthetic (containing chloroplasts) and nonphotosynthetic (devoid of chloroplasts) tissues was used as a basis to study cell specific gene expression of the cauliflower mosaic virus 35S promoter (CaMV), the chlorophyll a,b/binding protein promoter (cab) and small subunit of ribulose bisphosphate carboxylase promoter (rbcS), each fused to the gene encoding the enzyme beta-glucuronidase. Protoplasts from transgenic plants, each containing one of the three specific constructs, were subjected to flow analysis and sorting to separate cells from photosynthetic and nonphotosynthetic tissue types. Expression of the CaMV gene fusion was found in both cell types, while the expression of the photosynthetic (rbcS and cab) gene fusions occurred predominantly in protoplasts isolated from photosynthetic tissue. A similar pattern of expression was observed in wild-type protoplasts transfected with the same gene fusions. Photooxidation induced by the herbicide Norflurazon had no effect on the expression of the CaMV gene fusion, but reduced the level of expression of both the cab and rbcS gene fusions in both the transgenic and the transfected protoplasts.