Cytokinins in tobacco and wheat chloroplasts, occurrence and changes due to light/dark treatment

Although cytokinins (CKs) affect a number of processes connected with chlor oplasts, it has never been rigorously proven that chloroplasts contain CKs. We isolated intact chloroplasts from tobacco (Nicotiana tabacum L. cv SR1) and wheat (Triticum aestivum L. cv Ritmo) leaves and determined their CKs by liquid chromatography/tandem mass spectroscopy. Chloroplasts from both s pecies contained a whole spectrum of CKs, including free bases (zeatin and isopentenyladenine), ribosides (zeatin riboside, and isopentenyladenosine), ribotides (isopentenyladenosine-5'-monophosphate, zeatin riboside5'-monoph osphate, and dihydrozeatin ribeside-5'-monophosphate), and N-glucosides (ze atin- N-9-glucoside, dihydrozeatin-N-9-glucoside, zeatin-N-7-glucoside and isopentenyladenine-N-glucosides). In chloroplasts there was a moderately hi gher relative amount of bases, ribosides, and ribotides than in leaves, and a significantly increased level of N-9-glucosides of zeatin and dihydrozea tin. Tobacco and wheat chloroplasts were prepared from leaves at the end of either a dark or light period. After a dark period; chloroplasts accumulat ed more CKs than after a light period. The differences were moderate for fr ee bases and ribosides, but highly significant for glucosides. Tobacco chlo roplasts from dark-treated leaves contained zeatin riboside-O-glucoside and dihydrozeatin riboside-O-glucoside, as well as a relatively high CK oxidas e activity. These data show that chloroplasts contain a whole spectrum of C Ks and the enzymatic activity necessary for their metabolism.