Cytokinins in Tobacco and Wheat Chloroplasts. Occurrence and Changes Due to Light/Dark Treatment

Abstract

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.Cytokinins (CKs) regulate a number of growth and developmental processes in plants, including activation of cell division, stimulation of growth of axillary buds (suppression of apical dominance), inhibition of root growth, and suppression of senescence (for reviews, see Binns, 1994; Mok and Mok, 1994). CK-suppressed senescence was first reported by Richmond and Lang (1957) in detached Xanthium leaves kept in the dark. CKs were shown to maintain protein synthesis and to prevent chlorophyll degradation. Numerous subsequent studies initiated by the pioneering work of Mothes (1960) have shown that CKs also affect many other processes connected with chloroplasts. Exogenous CKs stimulate de-etiolation, i.e. the transition of etioplasts into chloroplasts in detached leaves and cell cultures (for reviews, see Parthier, 1979;Reski, 1994).CKs affect the abundance of transcripts and proteins encoded both by nuclear and plastid genomes, the most notable being genes coding for the small subunit of Rubisco and chlorophyll a/b binding protein (for reviews, see Link, 1988;Parthier, 1989). The mechanism by which this occurs has not been completely resolved, but there is evidence for translational and posttranslational control (Link, 1988). This control may be mediated by polyribosome formation (Ohya and Suzuki, 1998), modification of the secondary structure of poly(A ϩ ) RNA (Jackowski et al., 1987), phosphorylation of the ribosomal proteins (Yakovleva et al., 1992), or regulation of the activi...

Cytokinin-Deficient Transgenic Arabidopsis Plants Show Multiple Developmental Alterations Indicating Opposite Functions of Cytokinins in the Regulation of Shoot and Root Meristem Activity

“…It has been shown that specific spectra of cytokinin metabolites occur extracellularly as well as in subcellular compartments such as chloroplasts (Faiss et al, 1997;Benková et al, 1999). Sequence analyses of AtCKX proteins predict different subcellular localizations (see below), and the analyses described above of AtCKX overexpressers indicated that the site of enhanced cytokinin degradation might be relevant to the expression of the cytokinin deficiency syndrome.…”

Cytokinin Stimulates Chloroplast Transcription in Detached Barley Leaves

“…Exogenously applied cytokinins delay senescence of detached leaves and keep chloroplasts photosynthetically active longer than in control leaves not treated with cytokinins (Romanko et al, 1969). Chloroplasts harbor enzymes for the biosynthesis of cytokinins and contain a set of natural cytokinins, including free bases, ribosides, ribotides, and N-glucosides (Benkova et al, 1999;Kasahara et al, 2004;Polanska et al, 2007). The developmental and/or metabolic state of plastids influences the response of leaves to exogenous cytokinins (Kulaeva et al, 2002).…”

Cytokinin oxidase or dehydrogenase?. Mechanism of cytokinin degradation in cereals

“…If the suggested electron pathway were true, it would imply that CKX is located on biological membranes and utilizes an intramolecular electron transfer mechanism similar to succinate dehydrogenase (EC 1.3.5.1), a typical Fe-S flavoprotein. In this context, it is interesting that recent observations of tobacco chloroplasts from dark-treated leaves showed that they contained zeatin riboside-O-glucoside and dihydrozeatin riboside-O-glucoside, as well as a relatively high CKX activity [34].…”