Development and Use of Chlorotetracycline Fluorescence as a Measurement Assay of Chloroplast Envelope-Bound Mg2+

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Abstract
Experiments were conducted to develop chlorotetracycline (CTC) fluorescence as an assay of Mg2+ bound to the envelope of the intact chloroplast. This assay technique has been widely used to measure envelope associated divalent cations in animal cells and subcellular systems, but has not been used with chloroplasts. Chloroplast envelope-associated Mg2+ was altered by pretreatment with Mg2+ and divalent cation chelating agents and by additions of Mg2+ to the CTC assay medium. Results indicated that for a given chloroplast preparation, relative changes in envelope-associated Mg2+ can be effectively monitored with CTC fluorescence. It was concluded that the limitations of this assay system are: (a) chlorophyll strongly quenches CTC fluorescence signal, so a constant chlorophyll concentration must be maintained, (b) measurements must be made quickly, and (c) use of the technique to compare different chloroplast preparations may not be valid. Studies with 28Mg2+ confirmed our interpretation of the fluorescence results, and also suggested that the chloroplast envelope is fairly impermeable to Mg2+. It was concluded that changes in Mg2+ associated with the chloroplast due to incubation of plastids in solutions containing up to 5 millimolar Mg2+ may be exclusively due to increased envelope-associated Mg2+. The CTC assay was used in experiments to demonstrate that increases in chloroplast envelope-associated Mg2+ inhibit photosynthetic capacity. This inhibiton can be partially overcome by the presence of K+ in the photosynthetic reaction media.