Although cytokinins (CKs) affect a number of processes connected with chloroplasts, 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 species contained a whole spectrum of CKs, including free bases (zeatin and isopentenyladenine), ribosides (zeatin riboside, and isopentenyladenosine), ribotides (isopentenyladenosine-5'-monophosphate, zeatin riboside-5'-monophosphate, and dihydrozeatin riboside-5'-monophosphate), and N-glucosides (zeatin-N(9)-glucoside, dihydrozeatin-N(9)-glucoside, zeatin-N(7)-glucoside, and isopentenyladenine-N-glucosides). In chloroplasts there was a moderately higher relative amount of bases, ribosides, and ribotides than in leaves, and a significantly increased level of N(9)-glucosides of zeatin and dihydrozeatin. Tobacco and wheat chloroplasts were prepared from leaves at the end of either a dark or light period. After a dark period, chloroplasts accumulated more CKs than after a light period. The differences were moderate for free bases and ribosides, but highly significant for glucosides. Tobacco chloroplasts from dark-treated leaves contained zeatin riboside-O-glucoside and dihydrozeatin riboside-O-glucoside, as well as a relatively high CK oxidase activity. These data show that chloroplasts contain a whole spectrum of CKs and the enzymatic activity necessary for their metabolism.

Institute of Biophysics Academy of Sciences of the Czech Republic Královopolská 135 61265 Brno Czech Republic

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Arnon DI. Plant Physiol. 1949;24:1–15. PubMed PMC

Bieleski RL. The problem of halting enzyme action when extracting plant tissues. Anal Biochem. 1964;9:431–442. PubMed

Binns AN. Cytokinin accumulation and action: biochemical, genetic and molecular approaches. Annu Rev Plant Physiol Plant Mol Biol. 1994;45:173–196.

Bradford M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–254. PubMed

Chattfield JM, Armstrong DJ. Cytokinin oxidase from Phaseolus vulgaris callus tissue: enhanced in vitro activity of the enzyme in the presence of copper-imidazole complexes. Plant Physiol. 1987;84:726–731. PubMed PMC

Chen Ch-M Cytokinin biosynthesis and interconversion. Physiol Plant. 1997;101:665–673.

Davey JE, Van Staden J. Cytokinins in spinach chloroplasts. Ann Bot. 1981;48:243–246.

Fusseder A, Ziegler P. Planta. 1988;173:104–109. PubMed

Gregory EM, Fridovich I. Visualization of catalase on acrylamide gels. Anal Biochem. 1974;58:57–62. PubMed

Jackowski G, Jarmolowski A, Szweykowska A. Kinetin modifies the secondary structure of poly(A)RNA in cucumber cotyledons. Plant Sci. 1987;52:67–70.

Jameson PE. Cytokinin metabolism and compartmentation. In: Mok DWS, Mok M, editors. Cytokinins: Chemistry, Activity, and Function. Boca Raton, FL: CRC Press; 1994. pp. 113–128.

Jayabaskaran C, Kuntz M, Guillemant P, Weil JH. Plant Physiol. 1990;92:136–140. PubMed PMC

Jensen RG, Bassham JA. Photosynthesis by chloroplasts. Proc Natl Acad Sci USA. 1966;56:1095–1101. PubMed PMC

Kamínek M. Evolution of tRNA and origin of two positional isomers of zeatin. J Theor Biol. 1974;48:489–492. PubMed

Latzko E, Gibbs M. Distribution and activity of enzymes of the reductive pentose phosphate cycle in spinach leaves and in chloroplasts isolated by different methods. Z Pflanzenphysiol. 1968;59:184–194.

Link G. Photocontrol of plastid gene expression. Plant Cell Environ. 1988;11:329–338.

Machác̆ková I, Krekule J, Eder J, Seidlová F, Strnad M. Cytokinins in photoperiodic induction of flowering in Chenopodium species. Physiol Plant. 1993;87:160–166.

Mok DWS, Mok M, eds (1994) Cytokinins: Chemistry, Activity, and Function. CRC Press, Boca Raton, FL

Mothes K. Über das Altern des Blätter und die Möglichkeitbihrer Wiederverjüngung. Naturwissenschaften. 1960;47:337–351.

Motyka V, Kamínek M. Cytokinin oxidase from auxin- and cytokinin-dependent callus cultures of tobacco (Nicotiana tabacum L.) J Plant Growth Regul. 1994;13:1–9.

Ohya T, Suzuki H. Cytokinin-promoted polyribosome formation in excised cucumber cotyledons. J Plant Physiol. 1988;133:295–301.

Parthier B. The role of phytohormones (cytokinins) in chloroplast development. Biochem Physiol Pflanzen. 1979;174:173–214.

Parthier B. Hormone-induced alterations in plant gene expression. Biochem Physiol Pflanzen. 1989;185:289–314.

Prinsen E, Kamínek M, Van Onckelen HA. Cytokinin biosynthesis: a black box? Plant Growth Regul. 1997;23:3–15.

Prinsen E, Redig P, Van Dongen W, Esmans EL, Van Onckelen HA. Quantitative analysis of cytokinins by electrospray tandem mass spectrometry. Rapid Commun Mass Spectrom. 1995;9:948–953.

Redig P, Schmülling T, Van Onckelen H. Analysis of cytokinin metabolism in ipt transgenic tobacco by liquid chromatography-tandem mass spectrometry. Plant Physiol. 1996;112:141–148. PubMed PMC

Reski R. Plastid genes and chloroplast biogenesis. In: Mok DWS, Mok M, editors. Cytokinins: Chemistry, Activity, and Function. Boca Raton, FL: CRC Press; 1994. pp. 179–195.

Reski R, Abel WO. Analyzing cytokinins and plastid differentiation at the molecular level: interference with genome-plastome interactions, light quality, developmental state and endogenous oscillators. In: Kamínek M, Mok DWS, Zaz̆ímalová E, editors. Physiology and Biochemistry of Cytokinins in Plants. The Hague, The Netherlands: SPB Academic Publishing; 1992. pp. 255–260.

Richmond AE, Lang A. Effect of kinetin on protein content and survival of detached Xanthium leaves. Science. 1957;125:650–651. PubMed

Romanko EG, Selivankina SY, Moshkov IE, Novikova GV. Effect of cytokinin-binding proteins isolated from chloroplasts on transcription. Fiziol Rast. 1986;33:1078–1083.

Romanov GA. Cytokinins and tRNAs: a hypothesis on their competitive interaction via specific receptor proteins. Plant Cell Environ. 1990;13:751–754.

Simcox PD, Reid EE, Canvin DT, Dennis DT. Enzymes of the glycolytic and pentose phosphate pathways in proplastids from the developing endosperm of Ricinus communis L. Plant Physiol. 1977;59:1128–1132. PubMed PMC

Suzuki T, Kawano S, Sakai A, Fuji M, Kuroiwa H, Nakamura H, Kuroiwa T. J Cell Sci. 1992;103:831–836.

Swaminathan S, Bock RM. Subcellular localization of cytokinins in transfer ribonucleic acid. Plant Physiol. 1977;59:558–563. PubMed PMC

Taller BJ. Distribution, biosynthesis and function of cytokinins in tRNA. In: Mok DWS, Mok M, editors. Cytokinins: Chemistry, Activity, and Function. Boca Raton, FL: CRC Press; 1994. pp. 101–112.

Teyssandier de la Serve B, Axelos M, Peaud-Lenoel C. Cytokinins modulate the expression of genes encoding the protein of the light-harvesting chlorophyll a/b complex. Plant Mol Biol. 1985;5:155–163. PubMed

Tolbert NE (1974) Isolation of subcellular organelles of metabolism on isopycnic sucrose gradients. In S Fleischer, L Packer, eds, Methods in Enzymology, Vol 31. Academic Press, New York, pp 734–746 PubMed

Witters E, Vanhoutte K, Dewitte W, Machác̆ková I, Benková E, Van Dongen W, Esmans EL, Van Onckelen HA. Analysis of cyclic nucleotides and cytokinins in minute plant samples using phase-system switching capillary ion-spray LC-MSMS. Phytochem Anal. 1999;10:143–148.

Yakovleva LA, Klueva NY, Kulaeva NO. Phosphorylation of ribosome proteins as a mechanism of phytohormone regulation of protein synthesis in plants. In: Kamínek M, Mok DWS, Zaz̆ímalová E, editors. Physiology and Biochemistry of Cytokinins in Plants. The Hague, The Netherlands: SPB Academic Publishing; 1992. pp. 169–172.

Zhang HM, Huang H, Liu Y. Isolation and purification of cytokinin-binding protein from wheat chloroplasts. Acta Bot Sinica. 1991;33:744–749.

Zhang HM, Liu Y, Shen YG. Properties of a cytokinin-binding protein from wheat chloroplasts. Acta Phytophysiol Sinica. 1996;22:27–32.

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