The role of adenine in the synthesis of cytokinins in tomato plants and in cell-free root extracts

The incorporation of labelled adenine into cytokinin-like compounds was investigated in intact tomato plants, decapitated tomato roots and cell-free root extracts. In all three cases evidence was found for the incorporation of adenine into endogenous cytokinins. In intact plants and decapitated root systems no evidence was found for the incorporation into cytokinin nucleotides. Cytokinin bases and nucleosides were however, labelled. In the cell-free root extract there was some evidence for the incorporation of labelled adenine into cytokinin nucleotides. This suggests that the biosynthetic process may be strictly compartmentalized. The present results provide no evidence for the relative importance of cytokinin nucleotides in the biosynthetic process. What is clear is that the rate of adenine incorporation into cytokinins is extremely low and that only a small proportion of the cytokinins which became labelled were exported to the shoot via the root exudate.The financial support of the CSIR/Israel Collaborative Programme is gratefully acknowledged.     

<Emphasis Type="Italic">Rhodococcus fascians</Emphasis>: Shoot Proliferation without Elevated Cytokinins?

In order to determine whether the disease symptoms caused by virulent strains of Rhodococcus fascians are due to increased cytokinin activity in infected tissues, germinating peas (Pisum sativum cv Novella) were inoculated with either a virulent strain or a nonvirulent strain of Rhodococcus fascians. The nonvirulent strain lacked both the ipt gene and the putative cytokinin oxidase/dehydrogenase homologue, fas5. Control peas were not inoculated. Twelve cytokinins were isolated from pea shoots 3, 6 and 9 days post-inoculation. Within 6 days of inoculation the levels of cytokinin free bases, ribosides, O-glucosides and nucleotides were decreased in shoots inoculated with the virulent strain, and were increased in shoots inoculated with the nonvirulent strain relative to the uninoculated control. The results are discussed with respect to the classic Skoog and Miller (1965) model of organogenesis and to the possible involvement of the plant cytokinin oxidase/dehydrogenase during infection by virulent strains of R. fascians.     

The effect of auxin concentration on cytokinin stability and metabolism

The stability of [³H]zeatin riboside supplied to freshly excised tobacco pith explants was found to be inversely related to -naphthaleneacetic acid concentration in the incubation medium. At higher concentrations of -naphthaleneacetic acid greater breakdown of [³H]zeatin riboside was indicated by higher levels of degradative metabolites (adenine, adenosine and adenosine nucleotides) formed. This auxin effect on cytokinin metabolism appears to be mediated, at least in part, through cytokinin oxidase. The results of in-vitro assays carried out with partially purified enzyme from corn kernels substantiale this conclusion. These findings are discussed in relation to recent observations of auxin and cytokinin levels in crown-gall tumours with altered morphology.Abbreviations FPLC fast protein liquid chromatography - HPLC high-performance liquid chromatography - IP isopentenyladenine - NAA naphthaleneacetic acid - ZR zeatin riboside     

细胞分裂素合成基因ipt研究进展(综述)

异戊烯基转移酶是细胞分裂素生物合成第一步的催化酶,也是限速酶。其编码基因ipt已被克隆,运用生物信息学方法,在拟南芥中鉴定出与微生物同源的编码异戊烯基转移酶的基因家族,推测这些基因可能存在特殊时空表达来调控细胞分裂素的合成途径。本文着重介绍ipt在细胞分裂素合成中的作用和研究进展。     

Analysis of gametophytic development in the moss,Physcomitrella patens,using auxin and cytokinin resistant mutants

Mutants altered in their response to auxins and cytokinins have been isolated in the moss Physcomitrella patens either by screening clones from mutagenized spores for growth on high concentrations of cytokinin or auxin, in which case mutants showing altered sensitivities can be recognized 3–4 weeks later, or by non-selective isolation of morphologically abnormal mutants, some of which are found to have altered sensitivities. Most of the mutants obtained selectively are also morphologically abnormal. The mutants are heterogeneous in their responses to auxin and cytokinin, and the behaviour of some is consistent with their being unable to make auxin, while that of others may be due to their being unable to synthesize cytokinin. Physiological analysis of the mutants has shown that both endogenous auxin and cytokinin are likely to play important and interdependent roles in several steps of gametophytic development. Although their morphological abnormalities lead to sterility, genetic analysis of some of the mutants has been possible by polyethyleneglycol induced protoplast fusion.Abbreviations NTG N-methyl-N-nitro-N-nitrosoguanidine - NAA 1-naphthalene acetic acid - 2,4D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - IAP 6-( ²isopentenyl) aminopurine - NAR NAA resistant mutants - BAR BAP resistant mutants     

On the biogenesis of cytokinins in Lactobacillus acidophilus ATCC 4963

The biosynthesis of free cytokinins in the mevalonic acid auxotrophic Lactobacillus acidophilus , ATCC 4963 has been investigated. After a short pulse labelling with [¹⁴C]-mevalonic acid the labelled free cytokinins of bacteria and media and the labelled cytokinin-nucleotide moiety of tRNA and oligonucleotides were determined and compared. tRNA is the main precursor for cytokinin production. Bacteria previously starved for mevalonic acid showed the presence of at least one additional cytokinin precursor. A fraction of oligonudeotides shows rapid incorporation of ¹⁴C and contains labelled cytokinin nucleotides. There are no indications for a direct isopentenylation of adenine, adenosine or its phosphate derivatives.     

Factors affecting adventitious bud induction in Pinus elliottii (Engelm.) embryos cultured in vitro

Embryos of slash pine (Pinus elliottii Engelm.) were induced to form adventitious buds when placed in culture on nutrient media supplemented with cytokinin. Buds were induced on media containing Risser & White major salts. The high content in nitrogen of Murashige & Skoog formulation seems to be deleterious for this in vitro system, since morphogenic responses were only promoted when nitrogen concentration was drastically reduced in the macronutrient formulation. Factors such as concentration of cytokinin (6-benzyladenine) and time and method of exposure (liquid or solid induction medium) strongly influenced bud formation and development. The greatest number of buds and shoots were obtained from 22.0 M cytokinin, but these shoots showed less and slower development than those induced with low dosages of cytokinin. The presence of naphthaleneacetic acid in combination with cytokinin in the induction medium decreased the frequency of bud formation.Abbreviations (BA) 6-benzyladenine - (NAA) 1-naphthaleneacetic acid     

A comparative ultrastructural study of pollen development in Arabidopsis thaliana ecotype Columbia and male-sterile mutant apt1-3

Adenine phosphoribosyltransferase (APT) catalyzes the conversion of adenine and cytokinin bases to the corresponding nucleotides. An Arabidopsis thaliana mutant lacking the major APT isoform, APT1, is male sterile due to defects soon after meiosis. We have now used electron microscopy to define the effects of APT1 deficiency on pollen development to determine whether the changes might be attributed to adenine or cytokinin metabolism. Changes were observed in mutant anthers in both tapetal and pollen mother cells prior to meiosis with additional defects found at later stages, in both compartments. Principal changes include altered lipid accumulation in the tapetal cells, changes in pollen cell wall development, and a loss of synchrony in the development of the tapetum and microspores. Taken together our results suggest that APT1 deficiency causes a general metabolic decrease in energy metabolism, due to the lack of adenine recycling into adenylate nucleotides, which ultimately leads to pollen abortion. The early onset of meiosis in the mutant may be associated with altered cytokinin metabolism.     

Functional characterisation of a cytokinin oxidase gene DSCKX1 in Dendrobium orchid

Cytokinin oxidase plays an important role in the cytokinin regulatory processes. We have cloned a novel putative cytokinin oxidase, DSCKX1 (D endrobium Sonia cytokinin oxidase), by mRNA differential display from shoot apices of Dendrobium Sonia cultured in the presence of BA. The DSCKX1 gene appears to have three alternative splicing forms and its expression of DSCKX1 was induced in a tissue-specific manner by cytokinins. In transgenic orchid plants overexpressing DSCKX1, the elevated level of cytokinin oxidase activity was accompanied by a reduction of cytokinin content. These plants exhibited slow shoot growth with numerous and long roots in vitro. Their calli also showed decreased capability of shoot formation. Conversly, antisense transgenic plants showed rapid proliferation of shoots and inhibition of root growth combined with a higher endogenous cytokinin content than wild-type plants. Thus DSCKX1 appears to play an important role on cytokinin metabolism and the related developmental programmes in orchid.     

Influence of White Clover Mosaic Potexvirus Infection on the Endogenous Cytokinin Content of Bean

The cytokinin content in the primary leaves of bean (Phaseolus vulgaris) was monitored for 10 d after inoculation with white clover mosaic potexvirus. The cytokinins were isolated, purified, separated by high-performance liquid chromatography, and quantified by radioimmunoassay. The cytokinins detected at the time of inoculation (d 0) were: (a) the free bases, zeatin (Z), dihydrozeatin (DZ), and isopentenyladenine; (b) the riboside, DZ riboside (DZR); (c) the O-glucosides of DZ, DZR, and Z riboside; (d) the nucleotides, Z riboside-5′-monophosphate and isopentenyladenosine-5′-monophosphate; and (e) trace amounts of Z-9-glucoside and DZ-9-glucoside. During the 10 d after inoculation with white clover mosaic potexvirus, marked quantitative changes in this cytokinin profile were observed. The concentration of the free bases and DZR decreased, accompanied by an increase in the 9-glucosides and the nucleotides. Virus titer increased rapidly 3 d after inoculation, attaining a maximum level at d 5. This increase coincided with the increases in the 9-glucosides and the nucleotides. We propose that the decline in the cytokinin free bases and riboside may allow the increase of virus titer in bean and lead to the senescence of infected leaves.     

Rapid differentiation of tracheary elements in cultured explants of Jerusalem artichoke

the culture of Jerusalem artichoke (Helianthus tuberosus L.) tuber explants on filter paper discs moistened with liquid medium resulted in rapid and consistent xylem differentiation. The number of tracheary elements increased in discrete steps, the first at 48 h with a second at 56–58 h, following partially synchronous mitoses at 20 and 30 h. Factors favouring xylem cell differentiation were optimum levels of both an auxin and a cytokinin, low medium nitrogen concentrations, small volumes of medium, and high culture temperatures. A cell counting method employing Feulgen-stained nuclei and suitable for quantifyings small numbers of immature tracheary elements is described.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - NAA -naphthalene acetic acid - BAP benzylaminopurine - GA₃ gibberellic acid     

Cytokinin metabolism in Physcomitrella patens – differences and similarities to higher plants

Cytokinins play an important role in plant development and occur informs with different hormonal activity. As the nucleotide forms of cytokininsare considered to have little or no biological activity, the conversion ofcytokinin bases and ribosides to their nucleotides can contribute to the tuningof cytokinin activity in plant cells. Cytokinin metabolism was monitoredin vivo by feeding either radiolabelledisopentenyladenosine (³H-[9R]iP) or isopentenyladenine(³H-iP) to liquid grown chloronema tissue ofPhyscomitrellapatens (Hedw.) B.S.G. wild type. The riboside ³H-[9R]iPwas rapidly converted to ³H-iP, which was released into the culturemedium. The intracellular concentration of the ³H-iP was twice ashigh as extracellular. From the overall amount of ³H-iP about 95%were present in the medium. Cytokinin nucleotides occurred as tritiated mono-,di- and triphosphates of ³H-[9R]iP. When feeding the base³H-iP however, its main metabolic fate was degradation and nosignificant amounts of radiolabelled cytokinin nucleotides were detected. Forthe cytokinin metabolism in P. patens it is concludedthat,in contrast to higher plants nucleotides are mainly formed from ribosidesvia the adenosine kinase pathway and not byribophosphorylation of the cytokinin base via adeninephosphoribosyltransferase.     

Cytokinin biosynthesis and interconversion

To maintain hormone homeostasis, the rate of cytokinin biosynthesis, interconversion, and degradation is regulated by enzymes in plant cells. Cytokinins can be synthesized via direct (de novo) or indirect (tRNA) pathways. In the de novo pathway, a cytokinin nucleotide is synthesized from 5'-AMP and isopentenyl pyrophosphate; a key enzyme which catalyzes this synthesis has been isolated from plant tissues, slime mold, and some microorganisms. Studies on the in vitro synthesis of the isopentenyl side chain of cytokinin in tRNA demonstrated that the isopentenyl group was derived from mevalonate, and turnover of the cytokinin-containing tRNA may serve as a minor source of free cytokinins in plant cells. The interconversion of cytokinin bases, nucleosides and nucleotides is a major feature of cytokinin metabolism; and enzymes that regulate the interconversion have been identified. The N⁶-side chain and purine moiety of cytokinins are often modified and some of the enzymes involved in the modifications have been isolated. Most of the cytokinin metabolites have been characterized but very few enzymes regulating their metabolism have been purified to homogeneity. It remains a significant challenge to isolate plant genes involved in the regulation of cytokinin biosynthesis, interconversion and degradation.     

Endogenous cytokinin levels and metabolism of zeatin riboside in genetic tumour tissues and non-tumourous tissues of tobacco

The cytokinin content of stem tissues, primary genetic tumours (excised from 2-month-old plants) and 3-week-old in vitro cultured genetic tumour tissues derived from Nicotiana glauca (Grah.) × langsdorffii (Weinm.) and N. suaveolens (Lehm.) × langsdorffii (Weinm.) hybrids and stem tissues derived from 2-month-old N. suaveolens and N. langsdorffii plants has been analysed by radioimmunoassay. Stem tissues of tumour-prone hybrids contain high cytokinin levels (3–3.7 nmol g⁻¹). This increase is caused mainly by increased levels of cytokinin nucleotides, particularly those of zeatin nucleotide (0.5 nmol g⁻¹) in stem tissues of parent plants and 2.4 nmol g⁻¹ in stem tissues of hybrids). All other tissues contain lower cytokinin levels (0.7–1.7 nmol g⁻¹). Cytokinin bases and ribosides are major compounds in cultured tumour tissues while the nucleotides are dominant cytokinins in all freshly excised tissues from parent plants and their hybrids. In a separate study, the metabolic fate of supplied [³Hj-zeatin riboside. which is inactivated mainly by sidechain cleavage, has been studied. The results collectively suggest that cytokinins may be involved in tumourigenesis.