Pathogenesis of barley leaves by Helminthosporium teres I: Green island formation and the possible involvement of cytokinins

Infection sites/green islands were formed in host leaf tissue infected with drops of H. teres. They exhibited higher cytokinin-like activity, sugar and starch than their surrounding tissue and tissue under water drops. The cytokinin-like activity at the infection sites increased from 24 to 72 h of incubation. However, the cytokinin-like activity of the tissue surrounding the infection drops and the tissue under water drops fell from 24 to 72 h incubation. The culture filtrate extracts of the fungus also produced cytokinin-like activity which increased from 1 to 10 days incubation. Application of this culture filtrate extract evoked green island formation. Application of kinetin to host leaves duplicated the green island effect. Thin-layer chromatographic fractions of the tissue extracts and the culture filtrate extracts revealed that a major portion of cytokinin-like activity corresponded to zeatin and zeatin riboside. The presence of zeatin and zeatin riboside (both in tissue and culture filtrate extracts) was confirmed by high performance liquid chromatography. Increases in the amounts of cytokinin-like substances, particularly zeatin and zeatin riboside, attributed to pathogen influence are suggested to be involved in infection and pathogenicity of H. teres.     

Involvement of carbohydrates and cytokinins in pathogenicity ofHelminthosporium carbonum

Significant stimulation of the number of appressoria, penetration and colonization by conidia ofHelminthosporium carbonum occurred on decolorized maize leaves when exogenous carbohydrates and leaf leachates were added. Germination and germ tube length, however, did not exhibit appreciable differences on decolorized or non-decolorized maize leaves. Lower germination was recorded by leached conidia on decolorized leaves; while appressoria, penetration and colonization were absent. Addition of exogenous nutrients (sucrose>leaf leachates>yeast extract>glucose) enabled conidia to accomplish appressoria, penetration and colonization. Optimum levels for various nutrients observed were 2% (w/v) sucrose/glucose or 0.1% (w/v) yeast extract. Higher concentrations inhibited the infection stages of the pathogen. Depletion of host carbohydrates from green islands/infection sites adversely affect appressoria formation, penetration and colonization; and the loss of carbohydrates from the spore affects germination. Cytokinin-like activity at the infection site/green islands increased with the period of incubation of the host as compared to the surrounding tissue or tissue under water drops. The culture filtrate extracts ofH. carbonum recorded cytokinin-like activity which increased with growth of the fungus. TLC (thin layer chromatography) of cytokinin-like substances (tissue extract and culture filtrate) revealed major activity was confined to Rf zones 0.6 to 0.8 which co-chromatographed with zeatin and zeatin riboside. These substances increase at infection sites by virtue of which carbohydrates accumulate at these sites ensuring a continuous supply to the growing pathogen.     

Cytokinins in pathogenesis and disease resistance of Pyrenophora teres-barley and Dreschslera maydis-maize interactions during early stages of infection

Infection of Hordeum vulgare L. by Pyrenophora teresand of Zea mays by Dreschslera maydis were characterized by green island formation, higher cytokinin levels and accumulation of metabolites in the infected areas. Higher cytokinin concentrations of the order 6-Y,Y-dimethylallylaminopurine > zeatinriboside > zeatin >dihydrozeatinriboside were detected at infection sites of susceptible hosts. By virtue of these cytokinins, infection sites may be acting as metabolic sinks helping proliferation of the pathogen. Existence of translocatory sinks at infection zones was confirmed from autoradiographic studies,where, accumulation of labeled metabolites was prominent at infection sites of susceptible hosts. Upon infection the lower cytokinin levels of resistant hosts decreased further with progress of infection. In the infected resistant hosts the concentrations of zeatin/zeatinriboside were the maximum among the four identified cytokinins. The pathogen is also capable of secreting cytokinins as evident from quantification of cytokinins in culture filtrate extracts using HPLC. Since detached leaves were used in the experiments the increase/decrease of various cytokinin levels may be attributed to pathogen influence. The increase in cytokinin levels in the susceptible host may be aiding the growth of the pathogen on one hand, while the decrease in the infected resistant host may signal the host to activate defenses against a potential pathogen at the early stage of infection.This revised version was published online in October 2005 with corrections to the Cover Date.     

Phytohormones,Rhizobium mutants,and nodulation in legumes. VII. Identification and quantification of cytokinins in effective and ineffective pea root nodules using radioimmunoassay

Radioimmunoassays (RIA), employing antisera raised in rabbits against bovine serum albumin conjugates of zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine, were used to estimate levels of these cytokinins and their corresponding bases in samples of effective (nitrogen-fixing, Fix⁺), ineffective (nonnitrogen-fixing, Fix^–) pea root nodules and uninoculated roots. Assays were done on extracts of nodule tissue, 1–2 g fresh weight, or approximately 10 g fresh weight of root tissue, and high specific activity [³H]zeatin riboside was added during preparation of the extract for use as a recovery marker. Two different purification procedures were employed, each involving several purification steps. High performance liquid chromatography (HPLC) was the final step in both procedures. Fractions from HPLC were analyzed by RIA using the appropriate antiserum. The cytokinins, zeatin, zeatin riboside, dihydrozeatin riboside, isopentenyl adenine, and isopentenyladenosine were detected and quantified in nodule tissue, and similarly, in root tissue (with the exception of zeatin, which we were unable to quantify in root tissue). Cytokinin levels in nodule tissue were higher than those in root tissue. The major cytokinins detected in nodule tissue were zeatin, followed by zeatin riboside and then dihydrozeatin riboside. The levels of zeatin and zeatin riboside estimated in nodules in the present study by RIA were of the same order of magnitude, though tending to be a little higher, than values obtained previously by bioassay. Dihydrozeatin riboside was identified with confidence for the first time in nodule tissue. There was a general decline with age in cytokinin levels in nodules, but no major qualitative change in nodule cytokinins with age. For theRhizobium strains examined, the data did not indicate a clear correlation between nodule cytokinin levels and the effectiveness of nodules in nitrogen fixation.     

Cytokinin activity inCitrus jambhiri Lush. seedlings colonized by vesicular-arbuscular mycorrhizal fungi

Summary The influence of vesicular-arbuscular mycorrhizal symbiosis on cytokinin activity in Citrus jambhiri Lush, seedlings was investigated. C. jambhiri inoculated with cultures of Glomus caledonium (Nicol. and Gerd.), G. epigaeum (Dan. and Trappe), G. etunicatum (Becker and Gerd.), G. fasciculatum Thaxt. (Gerd, and Trappe) or G. mosseae (Nicol and Gerd.) was grown from seed for 105 days in a glasshouse. Cytokinin activity in roots and leaves of seedlings was analyzed using high-performance liquid chromatography, mass spectrometry and a bioassay. Seedling leaf tissue had greater cytokinin activity than root tissue. Zeatin, zeatin riboside, and their dihydro- and glucoside derivatives were isolated from leaves of 105-day-old seedlings inoculated with G. fasciculatum and G. mosseae. Cytokinin activity in roots and leaves was associated with differences in seedling total dry weight and vesicular-arbuscular mycorrhizal colonization. The ribose moiety and the saturated side chain apparently influence cytokinin transport and physiological activity in Citrus seedlings.     

Changes in cytokinin activity during flower development in Cosmos sulphureus Cav

Endogenous cytokinin activity was determined in the flowers of Cosmos sulphureus Cav. from bud emergence to full bloom using the soybean callus bioassay. Cytokinin activity was low early in flower development but increased prior to full bloom. In Sephadex LH-20 column chromatography of flower extracts, the cytokinins present co-eluted with zeatin, zeatin riboside and glucoside cytokinin. While the former two predominated prior to full bloom, cytokinin glucoside activity appeared to be at a maximum at full bloom. The possible relevance of these findings is discussed in relation to flower development.     

Identification of Cytokinins of Root Nodules of the Garden Pea, Pisum sativum L

Five cytokinin activities which induced soybean callus proliferation were detected in ethanol extracts of root nodules of the garden pea (Pisum sativum L., cv. Little Marvel). The most active factors among them were identified as zeatin and its riboside on the basis of their mobility on thin layer chromatography in three solvent systems. Smaller activities of zeatin ribotide, isopentenyladenine and its riboside were also detected. Cytokinin activity gradually decreased with the cultivation period, but no qualitative change in the active compounds was found.     

Osmotic pretreatment promotes axillary shooting from cauliflower curd pieces by acting through internal cytokinin level modifications

In vitro propagation of cauliflower has generally been achieved through axillary shoot proliferation of curd explants on Murashige and Skoog (MS) medium supplemented with an auxin and a cytokinin. Recently, it has been shown (Vandemoortele 1999) that a soaking in sucrose (-2 MPa for 24 h) of cauliflower curd explants, before culture without any growth regulator, also induced axillary branching. The later procedure avoids the phenomenon of hyperhydricity in the shoots formed. Axillary shooting obtained by the two methods appears to be mediated by modifications of internal cytokinin levels. The osmotic pretreatment did not influence auxin levels, but induced a zeatin and a zeatin riboside levels increase. Curd explants cultured with the usual procedure (on MS medium supplemented with 5 μmol/L BA and 0.5 μmol/L NAA) showed a zeatin and zeatin riboside levels increase of the same magnitude and a higher one for isopentenyl adenine and isopentenyl adenosine. The modification of the cytokinin status in the curd explants subjected to a short osmotic pretreatment thus should be less favourable for hyperhydricity.     

Cytokinins in developing fruits of Moringa pterigosperma Gaertn.

The existence of cytokinins both as a free form and as a constituent of t-RNA was investigated in young fruits of Moringa pterigosperma Gaertn. Purified methanol extract was separated into butanol insoluble and butanol soluble fractions. The cytokinin(s) in the butanol insoluble fraction was tentatively identified as zeatin nucleotide. The butanol soluble fraction contained cytokinins and was chromatographed on Sephadex LH-20 with 35% ethanol. The two active fractions from LH-20 column coincided with zeatin and zeatin riboside. Cytokinin per g tissue was high in early stages of fruit growth and then remained more or less constant. Alkaline phosphatase hydrolysis of t-RNA hydrolysate of fruit tissue showed considerable cytokinin activity.     

The stability and biological activity of cytokinin metabolites in soybean callus tissue

The activity, uptake and metabolism of cytokinin metabolites was determined in soybean (Glycine max (L.) Merr.) callus tissue. The following activity sequence was established: zeatin riboside (ZR)>zeatin (Z)>O-glucosides of Z, ZR and their dihydro derivatives>lupinic acid (an alanine conjugate of Z)>7- and 9-glucosides of Z which were almost inactive. The 7- and 9-glucosides and lupinic acid were taken up very slowly by the callus tissue and showed great metabolic stability, but some degradation to 7-glucosyladenine, 9-glucosyladenine and the 9-alanine conjugate of adenine occurred. Compared with its aglycone, O-glucosyl-ZR exhibited slow uptake and greatly enhanced stability but gas chromatographic-mass spectrometric analysis showed that appreciable amounts were hydrolyzed to ZR in the tissue. Both ZR and O-glucosyl-ZR were metabolised extensively, with adenine, adenosine, and adenine nucleotide(s) as the major metabolites. A diversity of minor metabolites of ZR were identified, including O-glucosides, lupinic acid and dihydrolupinic acid. The metabolism of ZR was suppressed by 3-isobutyl-1-methylxanthine. When compared with the soybean callus line normally used for cytokinin bioassays (cv. Acme, cotyledonary callus), related callus lines exhibited greatly differing growth responses to cytokinin: however, these were not reflected in marked differences in metabolism.Abbreviations GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - LA lupinic acid - OGZR O--D-glucopyranosylzeatin riboside - TLC thin-layer chromatography - IMX 3-isobutyl-1-methylxanthine - Z zeatin - ZR zeatin riboside     

Isolation and identification of substances inducing formation of tracheary elements in cultured carrot-root slices

Data are presented on the cytokinin status of seeds and seed components, at different stages of development in Phaseolus coccineus L., as determined with the soybean callus growth bioassay: A change in cytokinin types according to developmental stage occurred: from biologically very active less polar types (zeatin=Z) at early stages to more polar types (zeatin glucoside=Z₉G and zeatin riboside=Zr), with relatively low biological activity, at intermediate and late stages of seed development: When cytokinins were analyzed separately in embryos (embryo proper) and suspensors at two embryonic stages: heart-shaped (A) and middle cotyledonary embryos (stage B) respectively, it was found that: i) at stage A, the suspensor showed cytokinin activity at the level of Z, 2iPA (2-isopentenyladenosine) and Zr, whereas more polar cytokinins (Z₉G, Zr) were present in the embryo; ii) at stage B, when the embryo seems to become autonomous for cytokinin supply, there was a relative abundance of active cytokinins (Z, 2iPA) in the embryo to which Z₉G activity in the suspensor corresponded. It is concluded that the suspensor plays an essential role in embryogenesis by acting as a hormone source to the early embryo.Abbreviations GA gibberellic acid - 2iPA 2-isopentenyladenosine - Stage A heart-shaped embryo - siage B middle cotyledonary embryo - Z zeatin - Z₉G zeatin glucoside - Zr Zeatin riboside     

The control of bud dormancy in potato tubers. Measurement of the seasonal pattern of changing concentrations of zeatin-cytokinins

A radioimmunoassay, combined with high-performance liquid chromatography, has been used to analyse the zeatin-type cytokinins of potato (Solanum tuberosum L. cv. Majestic) tubers and tuber buds throughout growth and storage. During tuber growth, zeatin riboside was the predominant cytokinin detected in all tissues. Immediately after harvest, the total cytokinin concentration fell dramatically in the storage tissue, largely as a consequence of the disappearance of zeatin riboside. During storage, levels of cytokinins in the storage tissue remained relatively constant, but increased in the tuber buds. In the buds of tubers stored at 2°C there was a 20-to 50-fold increase in total cytokinin over six weeks, coinciding with the natural break of innate dormancy. At 10°C the rise in the level of bud cytokinins was slower, correlating with the longer duration of innate dormancy. Injecting unlabelled cytokinins into tubers in amounts known to induce sprouting gave rise to increases in cytokinin concentrations in the buds of the same order as the increase associated with the natural break of dormancy. Metabolism of injected cytokinins was greater in non-dormant than in dormant tubers. The roles of cytokinin concentration and the sensitivity of the buds to cytokinin in the control of dormancy are discussed.Abbreviations CK cytokinin - FW fresh weight - HPLC high-performance liquid chromatography - RIA radioimmunoassay - tio⁶ade 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-purine=zeatin - tio⁶adeglc⁹ 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-glucopyranosyl purine=zeatin-9-glucoside - tio⁶ado 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-ribofuranosyl purine=zeatin riboside - tio⁶ado-[³H]-diol a radioactive derivative of zeatin riboside, synthesised by periodate-oxidation followed by [³H]NaBH₄-reduction - tio⁶AMP 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-5-phosphoribofuranosyl purine=zeatin riboside 5-monophosphate - t(ioglc⁴)⁶ade 6-(4-O--D-glucopyranosyl-3-methylbut-trans-2-enylamino)-purine=zeatin-O-glucoside     

Cytokinin Activity in Lupinus albus L: IV. Distribution in Seeds

Endogenous levels of cytokinin activity were examined in Lupinus albus L. seed at intervals of 2 weeks after anthesis using the soybean callus bioassay. High levels of cytokinin activity per gram seed material were present in the seeds at 2, 4, and 6 weeks after anthesis. The cytokinin activity per gram seed material was low at 8 and 10 weeks after anthesis. Cytokinin activity associated with each seed was greatest at 6 weeks after anthesis. The majority of the activity in the seeds at 4, 6, and 8 weeks after anthesis was in the endosperm. Cytokinin activity was also detected in the testas and embryos at 4, 6, 8, and 10 weeks, and the suspensors at 4 weeks. Column chromatography of extracts of the different seed fractions on Sephadex LH-20 indicated that the cytokinins present coeluted with zeatin, zeatin riboside, and the glucoside cytokinins. It is suggested that cytokinins are accumulated in the seeds and are stored in the endosperm mainly in the form of ribosides and glucosides of zeatin. The reduction in cytokinin activity in the seed coincides with the reduction in endosperm volume and embryo growth and suggests that these compounds are utilized during the course of seed maturation.     

Cytokinins of dryZea mays seed: Quantification by radioimmunoassay and gas chromatography-mass spectrometry

The endogenous cytokinins present in dryZea mays seed were determined using both radioimmunoassay and gas chromatography—mass spectrometry. Similar values for bases and ribosides were obtained by the two methods. The cytokinins present in embryo and endosperm were estimated separately using radioimmunoassay; similar levels of cytokinins were found in these two tissues. The major cytokinins detected on a whole-seed basis were dihydrozeatin riboside, O-glucosyldihydrozeatin riboside, zeatin 9-glucoside, zeatin, and the nucleotides of zeatin, dihydrozeatin, and isopentenyladenine. Cytokinin levels in the mature dry seed were considerably lower than cytokinin levels published in the literature for immature seed. Unexpected activity in the radioimmunoassays was detected in the wash from the DEAE cellulose column chromatography step. The compound(s) responsible for this activity did not have the solvent partitioning characteristics of a cytokinin base or riboside. They eluted as a single fraction following high-performance liquid chromatography on a Zorbax C8 column; this fraction showed no activity in theAmaranthus bioassay for cytokinins, but inhibited the activity of authentic zeatin riboside present at an optimal concentration.     

Zeatin-9-glucoside,a major endogenous cytokinin of Vinca rosea L. crown gall tissue

Cultured crown gall tissue of Vinca rosea L. was found to contain, in addition to the previously reported cytokinins zeatin, zeatin riboside, and the 0-glucosides of these two compounds, relatively high levels of zeatin-9-D-glucopyranoside. This is the first conclusive identification of an endogenous cytokinin 9-glucoside.Abbreviations GC gas chromatography - HPLC high-performance liquid chromatography - I.D. internal diameter - RFE rotary film evaporation - TLC thin layer chromatography - TMS trimethylsilyl - UV ultraviolet - Z zeatin - Z7G zeatin-7-glucoside - Z9G zeatin-9-glucoside - Z0G zeatin-0-glucoside - ZR zeatin riboside - ZR0G zeatin riboside-0-glucoside     

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     

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.     

Separation of Cytokinins from Danish Cabbage by Column Chromatography on Insoluble Polyvinylpyrrolidone

Chromatography of partially purified extracts of Danish cabbage (Brassica oleracea var. capitata L.) heads on polyvinylpyrrolidone columns using a weak phosphate buffer indicated a cytokinin complex containing nine components active in the modified Amaranthus betacyanin bioassay. Similar elution patterns were obtained with extracts of four different cabbage varieties but varietal differences occurred in the biological activity from each component peak. Paper and thin-layer chromatography indicated the presence of both zeatin and zeatin riboside in the extracts but the other cytokinin-active components were not identified. Separation of sweet-corn extracts by this technique indicated that most of the cytokinin activity occurred in the elution volume where zeatin and zeatin riboside would normally occur.     

Effect of thidiazuron,light fluence rates and kanamycin on in vitro shoot organogenesis from excised Rubus cotyledons and leaves

The relationship between genotype, tissue age and endogenous cytokinin levels on adventitious bud formation on Lachenalia leaf tissue were investigated. The genotypes studied, showed a variation in bud formation. The hybrid explants responded differently to factorial combinations of BA and NAA. The growth regulators could not substitute for the regeneration potential of the genotype. Tissue age had a pronounced effect on regeneration potential. Young tissue formed the largest number of buds. An interaction between tissue age and genotype was detected. Cytokinin levels in young leaf tissue were higher than in older tissue. In young tissue no relationship was observed between the cytokinin level and the number of buds formed. However, in older tissue it appears as if a relatively low endogenous cytokinin level enhanced bud formation.Abbreviations BA benzyladenine - NAA naphthalene-1-acetic acid - Z zeatin - ZR ribosylzeatin     

Cytokinin metabolism in nondividing and auxin-induced dividing explants ofHelianthus tuberosus L. Tuber tissue

Aqueous solutions of auxin (indole-3-acetic acid,-naphthalene acetic acid, or 2,4-dichlorophenoxyacetic acid) were active in inducing DNA synthesis and mitosis in prewashed tissue explants of mature Jerusalem artichoke tubers. Explants did not respond in this way to aqueous solutions of cytokinin (zeatin, zeatin riboside, 6-benzylaminopurine, or kinetin). The metabolism of [8-³H]zeatin riboside (ZR) was studied in non-dividing and auxin-induced synchronously dividing explants over the first 36 h of culture. ZR was taken up rapidly and to the same extent by both tissues. Sequential analysis of tissue extracts by thin-layer and high-performance-liquid chromatography identified zeatin nucleotide(s) (ZN), O-glucosyl zeatin riboside (OGZR), adenosine, and adenine nucleotide(s) (AN) as the principal metabolites in both tissues. The proportion of radio-activity due to ZR declined steadily and OGZR accumulated steadily at similar rates in both tissues. ZN was the major metabolite in both tissues at 12 h; thereafter ZN continued to accumulate in nondividing tissue, but its level declined in dividing tissue, and a corresponding increase in the levels of AN and adenosine was observed. These treatment differences in cytokinin metabolism were apparent at least 6 h before the onset of mitosis.