Altered cytokinin metabolism affects cytokinin, auxin, and abscisic acid contents in leaves and chloroplasts, and chloroplast ultrastructure in transgenic tobacco.

Cytokinins (CKs) are involved in the regulation of plant development including plastid differentiation and function. Partial location of CK biosynthetic pathways in plastids suggests the importance of CKs for chloroplast development. The impact of genetically modified CK metabolism on endogenous CK, indole-3-acetic acid, and abscisic acid contents in leaves and isolated intact chloroplasts of Nicotiana tabacum was determined by liquid chromatography/mass spectrometry and two-dimensional high-performance liquid chromatography, and alterations in chloroplast ultrastructure by electron microscopy. Ectopic expression of Sho, a gene encoding a Petunia hybrida isopentenyltransferase, was employed to raise CK levels. The increase in CK levels was lower in chloroplasts than in leaves. CK levels were reduced in leaves of tobacco harbouring a CK oxidase/dehydrogenase gene, AtCKX3. The total CK content also decreased in chloroplasts, but CK phosphate levels were higher than in the wild type. In a transformant overexpressing a maize beta-glucosidase gene, Zm-p60.1, naturally targeted to plastids, a decrease of CK-O-glucosides in chloroplasts was found. In leaves, the changes were not significant. CK-O-glucosides accumulated to very high levels in leaves, but not in chloroplasts, of plants overexpressing a ZOG1 gene, encoding trans-zeatin-O-glucosyltransferase from Phaseolus lunatus. Manipulation of the CK content affected levels of indole-3-acetic and abscisic acid. Chloroplasts of plants constitutively overexpressing Sho displayed ultrastructural alterations including the occasional occurrence of crystalloids and an increased number of plastoglobuli. The other transformants did not exhibit any major differences in chloroplast ultrastructure. The results suggest that plant hormone compartmentation plays an important role in hormone homeostasis and that chloroplasts are rather independent organelles with respect to regulation of CK metabolism.

Ectopic over-expression of the maize beta-glucosidase Zm-p60.1 perturbs cytokinin homeostasis in transgenic tobacco.

The activity of the phytohormone cytokinin depends on a complex interplay of factors such as its metabolism, transport, stability, and cellular/tissue localization. O-glucosides of zeatin-type cytokinins are postulated to be storage and/or transport forms, and are readily deglucosylated. Transgenic tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) plants were constructed over-expressing Zm-p60.1, a maize beta-glucosidase capable of releasing active cytokinins from O- and N3-glucosides, to analyse its potential to perturb zeatin metabolism in planta. Zm-p60.1 in chloroplasts isolated from transgenic leaves has an apparent K(m) more than 10-fold lower than the purified enzyme in vitro. Adult transgenic plants grown in the absence of exogenous zeatin were morphologically indistinguishable from the wild type although differences in phytohormone levels were observed. When grown on medium containing zeatin, inhibition of root elongation was apparent in all seedlings 14 d after sowing (DAS). Between 14 and 21 DAS, the transgenic seedlings accumulated fresh weight leading later (28-32 DAS) to ectopic growths at the base of the hypocotyl. The development of ectopic structures correlated with the presence of the enzyme as demonstrated by histochemical staining. Cytokinin quantification showed that transgenic seedlings grown on medium containing zeatin accumulate active metabolites like zeatin riboside and zeatin riboside phosphate and this might lead to the observed changes. The presence of the enzyme around the base of the hypocotyl and later, in the ectopic structures themselves, suggests that the development of these structures is due to the perturbance in zeatin metabolism caused by the ectopic presence of Zm-p60.1.

Three-dimensional reconstruction of anomalous chloroplasts in transgenic ipt tobacco.

Anomalies in the ultrastructure of chloroplasts, from transgenic ipt tobacco, overproducing endogenous cytokinins (CKs) were studied. Detailed analyses of CKs and their metabolites showed that Pssu-ipt tobacco contained enhanced contents of CKs both in leaves and in isolated chloroplasts. The role of CKs in the formation of anomalous structures is suggested. Pssu-ipt chloroplasts frequently formed the distinct peripheral reticulum with a system of caverns that often involved mitochondria and/or peroxisomes. Large crystalloids, which were found in chloroplasts of Pssu-ipt, occupied up to 16% of chloroplast volume. We suggested that the crystalloids were formed by LHC II aggregates. This was supported by analysis of the fluorescence emission spectra at 77 degrees K, chlorophyll a/b ratio, immunogold staining of the structures, and crystallographic unit size analysis.

Viability, ultrastructure and cytokinin metabolism of free and immobilized tobacco chloroplasts.

Cytokinins play a decisive role in regulation of plastid development and differentiation, but their metabolism in plastids is not known. Metabolic studies using intact chloroplasts are prevented by their instability once they are isolated from leaf cells. Chloroplasts of Nicotiana tabacum L. cv. Petit Havana SR1 were therefore immobilized into low-viscosity alginate. Their intactness was assessed by a glyceraldehyde-3-phosphate dehydrogenase assay which indicated that free chloroplasts totally disintegrated within 7 h, while more than 50% of immobilized chloroplasts remained intact after 24 h. The immobilization had no marked impact on ultrastructure and postponed final destruction. The metabolite profile was similar in free and immobilized chloroplasts after 4 h incubation with tritiated zeatin. Nevertheless, the yield of conversion products decreased twice in immobilized chloroplasts, which was probably the outcome of mass transfer limitations and/or the sorption to polysaccharide matrix.