Identification and quantitation by GC-MS of zeatin and zeatin riboside in xylem sap from rootstock and scion of grafted apple trees

Zeatin and zeatin riboside were identified by full-scan gas chromatography-mass spectrometry (GC-MS) in xylem sap of clonal apple rootstocks (M.27, M.9 and MM.106). These rootstocks exhibit a wide range of control over tree size when grafted to a common scion. The concentrations of zeatin and zeatin riboside were measured by GC-MS selected ion monitoring (SIM) in shoot xylem sap and root pressure exudate obtained from these rootstocks and from trees of Fiesta scion grafted onto the rootstocks. Zeatin was the predominant cytokinin in xylem sap from the dwarfing rootstocks, M.27 and M.9, while zeatin riboside was the predominant cytokinin in xylem sap from the more invigorating rootstock MM.106. Cytokinin concentrations (ng ml^–1) in root pressure exudate and shoot xylem sap, (i.e. from above the graft union in composite trees), increased with increasing vigour of the rootstock, irrespective of whether the plants were non-grafted rootstocks, or were composite plants of Fiesta scion grafted onto the rootstocks. Cytokinin content (ng shoot^–1) of shoot sap differed with rootstock; the more invigorating (MM.106) had greater amounts of cytokinins than the more dwarfing (M.9 and M.27) rootstocks. These results are discussed in relation to possible influences of roots on the growth of shoots via cytokinin supplies in the xylem sap.     

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.     

Effects of inoculum density,zeatin and sucrose on anthocyanin accumulation in a carrot suspension culture

Anthocyanin formation in a suspension culture of Daucus carota is induced by transfer from medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) to one lacking 2,4-D. The specific yields were strongly influenced by the inoculum density. Inoculum density altered the effect of zeatin concentration on anthocyanin accumulation. The in part by increasing the sucrose levels. It was inferred from the results that sucrose was exhausted at a low concentration of sucrose and at a high cell density, resulting in the decrease of yield of anthocyanin.     

The Roles of Phytohormones in Development as Studied in Transgenic Plants

The study of transgenic plants has greatly advanced our understanding of the control of development and metabolism. The ability to isolate and modify genes greatly extends the range of what is technically feasible. In the area of hormone biology, transgenic plants have helped to elucidate the pathways of synthesis, the metabolic control points, and the biological functions of the various phytohormones. This review covers the available genes that modulate the metabolism and perception of the phytohormones. One of the most significant conclusions coming out of transgenic plant work is the complex interaction among the different classes of phytohormones. For example, increasing the level of the auxin indole-3-acetic acid (IAA) in a plant has the secondary effect of inducing ethylene biosynthesis. This complication can be circumvented by combining transgenic plants modulating multiple hormones or through the use of available mutants. In this manner, transgenic plants have been utilized to unambiguously define the roles of auxin, cytokinin, and ethylene in the control of apical dominance. The power of transgenic plants as tools in hormone biology is perhaps best illustrated by work on ethylene. In this case, the modular characterization of genes led to elucidation of the biosynthetic pathway. Availability of the biosynthetic genes has permitted detailed analysis of the regulation of synthesis, definition of the role of ethylene in the control of several developmental processes, and the application of that knowledge for agricultural improvement.     

Effect of zeatin on the growth and indolyl-3-acetic acid and abscisic acid levels in maize roots

Elongation, indolyl-3-acetic acid (IAA) and abscisic acid (ABA) levels, – gas chromatography-mass spectrometry quantification –, in the elongating zone were analysed for maize ( Zea mays L., Cv. LG11) roots immersed in buffer solution with or without zeatin (Z). The effect of Z depends on the initial extension rate of roots. The slower growing roots are more strongly inhibited by Z (10⁻⁷−10₋₅ M ) and they show a greater increase in IAA and ABA content. When compared to the rapidly growing roots, the larger reactivity of the 'slow'ones cannot be attributed to a higher Z uptake as shown when using [¹⁴C]-Z. It is suggested that Z could regulate root elongation by acting on the IAA and/or ABA level. The comparative action of these two hormones is discussed.     

The role of cytokinins in responses to water deficit in tobacco plants over-expressing trans-zeatin O-glucosyltransferase gene under 35S or SAG12 promoters

The impact of water deficit progression on cytokinin (CK), auxin and abscisic acid (ABA) levels was followed in upper, middle and lower leaves and roots of Nicotiana tabacum L. cv. Wisconsin 38 plants [wild type (WT)]. ABA content was strongly increased during drought stress, especially in upper leaves. In plants with a uniformly elevated total CK content, expressing constitutively the trans -zeatin O-glucosyltransferase gene ( 35S::ZOG1 ), a delay in the increase of ABA was observed; later on, ABA levels were comparable with those of WT.
As drought progressed, the bioactive CK content in leaves gradually decreased, being maintained longer in the upper leaves of all tested genotypes. Under severe stress (11 d dehydration), a large stimulation of cytokinin oxidase/dehydrogenase (CKX) activity was monitored in lower leaves, which correlated well with the decrease in bioactive CK levels. This suggests that a gradient of bioactive CKs in favour of upper leaves is established during drought stress, which might be beneficial for the preferential protection of these leaves.
During drought, significant accumulation of CKs occurred in roots, partially because of decreased CKX activity. Simultaneously, auxin increased in roots and lower leaves. This indicates that both CKs and auxin play a role in root response to severe drought, which involves the stimulation of primary root growth and branching inhibition.     

Putative zeatin O‐glucosyltransferase OscZOG1 regulates root and shoot development and formation of agronomic traits in rice

As a ubiquitous reaction, glucosylation controls the bioactivity of cytokinins in plant growth and development.Here we show that genetic manipulation of zeatin-Oglucosylation regulates the formation of important agronomic traits in rice by manipulating the expression of OscZOG1 gene,encoding a putative zeatin O-glucosyltransferase. We found that OscZOG1 was preferentially expressed in shoot and root meristematic tissues and nascent organs. The growth of lateral roots was stimulated in the overexpression lines, but inhibited in RNA interference lines. In shoots, knockdown of OscZOG1 expression by RNA interference significantly improved tillering, panicle branching, grain number per panicle and seed size, which are important agronomic traits for grain yield. In contrast, constitutive expression of OscZOG1 leads to negative effects on the formation of the grain-yielding traits with a marked increase in the accumulation levels of cis-zeatin O-glucoside(c ZOG) in the transgenic rice plants. In this study,our findings demonstrate the feasibility of improving the critical yield-determinant agronomic traits, including tiller number, panicle branches, total grain number per panicle and grain weight by downregulating the expression level of OscZOG1. Our results suggest that modulating the levels of cytokinin glucosylation can function as a fine-tuning switch in regulating the formation of agronomic traits in rice.     

Regulation of cytokinin oxidase activity in tobacco callus expressing the T-DNA ipt gene

There are indications that the cytokinin content in transgenic tissues expressing the cytokinin biosynthetic ipt gene is under metabolic control, which prevents the accumulation of cytokinins to lethal levels. The objective of this study was to investigate the relationships between the content of endogenous cytokinins and the activity of cytokinin oxidase (which is believed to be a copper-containing amine oxidase, EC 1.4.3.6.) in ipt transgenic tobacco callus. In addition, the effect of exogenously applied N-benzyladenine (BA) on this relationship was examined. Endogenous cytokinin concentrations were measured in callus of Nicotiana tabacum L. cv. Petit Havana SRI transformed with the ipt of Agrobacterium tumefaciens under the control of a light-inducible promoter and in non-transformed tissue using LC-tandem mass spectrometry. The activity of cytokinin oxidase was estimated by measuring the conversion of [2,8-³H]N⁶-(Δ²-isopentenyl)adenine to [³H]adenine by enzyme preparations in vitro. The 14-day-old ipt-transformed callus contained a 25-fold higher amount of cytokinins as compared to the non-transformed tissue. Mainly zeatin- and dihydrozeatin-types of cytokinins (free bases, ribosides, nucleotides and O-glucosides) accumulated in the ipt transgenic tissue. The cytokinin pool of both ipt-transformed and non-transformed tissues consisted predominantly of cytokinins that are either resistant to cytokinin oxidase attack (nucleotides and O-glucosides of cytokinins and cytokinins bearing N⁶-saturated side chain) or have a low affinity for the enzyme (zeatin and its riboside). The former represented 71.6 and 74.8% and the latter 27.7 and 24.4% of the pool of endogenous cytokinins in ipt-transformed and non-transformed tissues, respectively. Enzyme preparations from ipt-transformed tissue exhibited 1.5-fold higher cytokinin oxidase activity compared with that observed in control tissues. Application of exogenous BA affected the total levels of cytokinins of the two tissue lines in different ways. The cytokinin content increased by 1.7- and 1.5-fold in ipt-transformed tissues 6 and 12 h after BA application, respectively, while it declined in the non-transformed control by 1.6- to 2.0-fold between 3 and 12 h after BA application. The increase in cytokinin content in the ipt callus is due to an increase of zeatin- and dihydrozeatin-type cytokinins (nucleotides, ribosides and free bases) leading to an enhanced accumulation of O-glucosides after 12 h. Following BA treatment, the cytokinin oxidase activity increased up to 1.8-fold in ipt-transformed and 1.6-fold in non-transformed tissues. The levels of isopentenyl-type cytokinins were near the detection limit; however, the enhancement of cytokinin oxidase activity after BA treatment in both tissue lines was correlated with the content of preferred substrate of the enzyme, N⁶-(Δ²-isopentenyl)adenosine.     

Anther-specific expression of ipt gene in transgenic tobacco and its effect on plant development

Isopentenyl transferase (ipt) gene from Agrobacterium tumefaciens T-DNA was placed under the control of a TA29 promoter which expresses specifically in anther. The chimeric TA29-ipt gene was transferred to tobacco plants. During flowering, mRNA of the ipt gene in the anthers of the transgenic plants accumulated and the level of iPA + iPs increased 3–4-fold in the leaves, petals, pistils, and stamens compared with those in the wild type plants. This cytokinin increase affected various aspects in development indicating that the alterations of endogenous cytokinin level by using anther-specific expression of the TA29-ipt gene affected morphology, floral organ systems and reproductivity of the transgenic plants.     

Metabolism of zeatin riboside in a hormone autonomous genetic tumour line of tobacco

The metabolic fate of externally applied [³H]-zeatin riboside ([9R]Z) was studied in a cultured genetic tumour line of Nicotiana glauca (Grah.) × N. langsdorffii (Weinm.), which grows on auxin and cytokinin free medium. Metabolism by 3.5-week-old tissues showed enhanced stability of supplied [9R]Z; unmetabolized [9R]Z accounted for 48.7 and 37.5% of extracted radioactivity following 8 and 24 h incubation, respectively; tissues of different ages (1–10 weeks following subculture) also indicated high cytokinin stability following 8 h incubation (unmetabolized [9R]Z accounted for 32.5–53.0% of extracted radioactivity). All analyses were performed by thin layer chromatography (TLC) and the results subsequently confirmed by high performance liquid chromatography (HPLC). Side-chain cleavage and modification of the purine ring were the major forms of metabolism; metabolites with an intact cytokinin moiety included zeatin (Z), [9R]Z nucleotides and glucosyl derivatives. Detailed analysis of metabolites carried out in the experiments using 3.5-week-old tissues indicated that both dihydro-derivatives as well as cis isomers of Z and [9R]Z were not formed. Adenine, adenosine and its nucleotide(s) were the main degradative metabolites; in 3.5-week-old tissues these metabolites accounted for about 5.9 and 7.8% (of ³H extracted) following 8 and 24 h incubation, respectively. In tissues of different ages (1–10 weeks following subculture), these metabolites accounted for about 7.6–22.9% of the extracted ³H. Some metabolites (zeatin, adenine and adenosine) were also detected in the staled incubation media. The observed high [9R]Z stability in this tissue may reflect low levels of cytokinin oxidase activity and/or some form of compartmentation.     

Morphological changes and hypomethylation of DNA in transgenic tobacco expressing antisense RNA of the S-adenosyl-l-homocysteine hydrolase gene

S-adenosyl-l-homocysteine hydrolase (SAHH) is a key enzyme in the regulation of intracellular methylation reactions. To investigate the role of SAHH in methylation reactions and morphogenesis in planta, we have made transgenic plants expressing antisense RNA of tobacco SAHH. The transgenic plants displayed distinct morphological changes including a floral homeotic change. We hypothesized that the changes were caused by increased levels of cytokinin. In those transgenic plants, we observed that a repetitive DNA sequence appeared less methylated than controls. We speculated that altered gene expressions by the hypomethylation of DNA might be involved in the changes.     

The expression of a heat-inducible chimeric gene in transgenic tobacco plants

Summary A chimeric gene under the control of the hsp70 promoter of Drosophila is heat regulated in roots, stems and leaves, but not in pollen of transgenic tobacco plants. For these and other parameters, it behaves similarly to plant heat-shock genes.     

Role of cytokinin and auxin in shaping root architecture: regulating vascular differentiation, lateral root initiation, root apical dominance and root gravitropism

BACKGROUND AND AIMS: Development and architecture of plant roots are regulated by phytohormones. Cytokinin (CK), synthesized in the root cap, promotes cytokinesis, vascular cambium sensitivity, vascular differentiation and root apical dominance. Auxin (indole-3-acetic acid, IAA), produced in young shoot organs, promotes root development and induces vascular differentiation. Both IAA and CK regulate root gravitropism. The aims of this study were to analyse the hormonal mechanisms that induce the root's primary vascular system, explain how differentiating-protoxylem vessels promote lateral root initiation, propose the concept of CK-dependent root apical dominance, and visualize the CK and IAA regulation of root gravitropiosm. KEY ISSUES: The hormonal analysis and proposed mechanisms yield new insights and extend previous concepts: how the radial pattern of the root protoxylem vs. protophloem strands is induced by alternating polar streams of high IAA vs. low IAA concentrations, respectively; how differentiating-protoxylem vessel elements stimulate lateral root initiation by auxin-ethylene-auxin signalling; and how root apical dominance is regulated by the root-cap-synthesized CK, which gives priority to the primary root in competition with its own lateral roots. CONCLUSIONS: CK and IAA are key hormones that regulate root development, its vascular differentiation and root gravitropism; these two hormones, together with ethylene, regulate lateral root initiation.     

Expression of the isopentenyl transferase gene is regulated by auxin in transgenic tobacco tissues

The isopentenyl transferase gene (ipt) fromAgrobacterium tumefaciens was isolated and introduced, via a disarmed binary vector, into tobacco using theAgrobacterium tumefaciens-mediated gene transfer system. The expression of theipt gene was monitored by RNA hybridization, western blotting and cytokinin analysis. The addition of auxin to the media rapidly reduced the level of cytokinins in the transgenic tissues and this was associated with a reduction in IPT mRNA and protein levels. It is concluded that the hormone auxin can regulate expression of a gene involved in biosynthesis of the second hormone cytokinin. Although exogenous benzyladenine did not directly affectipt gene expression, it did antagonize the effect of auxin on levels of cytokinins and IPT mRNA and protein.     

The effect of auxin on cytokinin levels and metabolism in transgenic tobacco tissue expressing an ipt gene

The ipt gene from the T-DNA of Agrobacterium tumefaciens was transferred to tobacco (Nicotiana tabacum L.) in order to study the control which auxin appears to exert over levels of cytokinin generated by expression of this gene. The transgenic tissues contained elevated levels of cytokinins, exhibited cytokinin and auxin autonomy and grew as shooty calli on hormone-free media. Addition of 1-naphthylacetic acid to this culture medium reduced the total level of cytokinins by 84% while 6-benzylaminopurine elevated the cytokinin level when added to media containing auxin. The cytokinins in the transgenic tissue were labelled with ³H and auxin was found to promote conversion of zeatin-type cytokinins to ³H-labelled adenine derivatives. When the very rapid metabolism of exogenous [³H]zeatin riboside was suppressed by a phenylurea derivative, a noncompetitive inhibitor of cytokinin oxidase, auxin promoted metabolism to adenine-type compounds. Since these results indicated that auxin promoted cytokinin oxidase activity in the transformed tissue, this enzyme was purified from the tobacco tissue cultures. Auxin did not increase the level of the enzyme per unit tissue protein, but did enhance the activity of the enzyme in vitro and promoted the activity of both glycosylated and non-glycosylated forms. This enhancement could contribute to the decrease in cytokinin level induced by auxin. Studies of cytokinin biosynthesis in the transgenic tissues indicated that trans-hydroxylation of isopentenyladenine-type cytokinins to yield zeatin-type cytokinins occurred principally at the nucleotide level.Abbreviations Ade adenine - Ados adenosine - BA 6-benzylaminopurine - C control - Con A concanavallin A - CP cellulose phosphate - IPT isopentenyl transferase - NAA 1-naphthylacetic acid - NP normal phase - NPPU N-(3-nitrophenyl)-N-phenylurea - RIA radioimmunoassay - RP reversed phase We wish to thank Dr. J. Zwar for supplying phenylurea derivitives.     

Variability of organ-specific gene expression in transgenic tobacco plants

Variability of expression of introduced marker genes was analysed in a large number of tobacco regenerants from anAgrobacterium-mediated transformation. In spite of standardization of sampling, considerable variation of GUS and NPTII expression was observed between individual transformants at different times of analysis and in different parts of the same plant. Organ-specificity of root versus leaf expression conferred by the par promoter from the haemoglobin gene ofParasponia andersonii in front of thegus gene showed a continuous spectrum. GUS expression in roots was found in 128 out of 140 plants; expression in leaves was found in 46 plants, and was always lower than in the corresponding roots. NPTII expression regulated by the nos promoter also showed a continuous spectrum. Expression levels were generally higher in roots than in leaves. Plants with high GUS expression in leaves showed high NPTII activity as well. A positive correlation between the level of NPTII expression and the numbers of integrated gene copies was noted. Chromosomal position effects and physiological determination are suggested as triggers for the variations. The transformed regenerated tobacco plants were largely comparable to clonal variants.     

Light-induced expression of ipt from Agrobacterium tumefaciens results in cytokinin accumulation and osmotic stress symptoms in transgenic tobacco

Cytokinins are plant growth regulators that induce shoot formation, inhibit senescence and root growth. Experiments with hydroponically grown tobacco plants, however, indicated that exogenously applied cytokinin led to the accumulation of proline and osmotin. These responses were also associated with environmental stress reactions, such as salt stress, in many plant species. To test whether increased endogenous cytokinin accumulation led to NaCl stress symptoms, the gene ipt from Agrobacterium tumefaciens, encoding isopentenyl transferase, was transformed into Nicotiana tabacum cv. SR-1 under the control of the light-inducible rbcS-3A promoter from pea. In high light (300 mol PPFD m^-2 s^-1), ipt mRNA was detected and zeatin/zeatin glucoside levels were 10-fold higher than in control plants or when transformants were grown in low light (30 mol PPFD m^-2 s^-1). High light treatment was accompanied by increased levels of proline and osmotin when compared to low light grown transformed and untransformed control plants. Elevated in planta cytokinin levels induced responses also stimulated by salt stress, suggesting either common or overlapping signaling pathways are initiated independently by cytokinin and NaCl, setting in motion gene expression normally elicited by developmental processes such as flowering or environmental stress.Abbreviations IPT isopentenyl, transferase - rbcS-3A gene encoding a small subunit protein (SSU) of Rubisco from Pisum sativum - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase