The possible involvement of cytokinins in the pathogenicity of Helminthosporium maydis

Green islands/infection sites recorded higher cytokinin activity than surrounding tissue as well as non-inoculated tissue. This activity in infected areas increased with time of incubation while in tissue surrounding the green islands and non-inoculated tissue, cytokinin activity decreased with time of incubation. The culture filtrate extracts of H. maydis had cytokinin activity which increased with growth of the fungus. Cytokinin activity of thin-layer Chromatographic fractions from tissue and culture filtrate extracts revealed that a major portion of the activity was confined to Rf zone 0.6 to 0.8 which co-chromatographed with zeatin and zeatin riboside. Presence of zeatin and zeatin riboside in tissue and culture filtrates was confirmed by high performance liquid chromatography. Cytokinin substances, such as zeatin and zeatin riboside, increase at infection sites with growth of the pathogen suggesting they may be involved in the pathogenicity of H. maydis on maize.     

Reduction of the number of nuclei per cell ofHelminthosporium euphorbiae by protoplast isolation

Helminthosporium euphorbiae is a pathogen of the weedEuphorbia heterophylla, which causes severe losses in soybean (Glycine max) crops. The fungus causes leaf loss and affects germination, making it a promising biocontrol agent for this weed. In order to start a breeding program for this species, four isolates were examined for number of nuclei in the conidia and hyphae and nuclear behavior at different cultivation stages. The conidia were multinucleated with about 20 nuclei per conidium, and 5 to 7 nuclei were observed in the hyphae compartments. The high number of nuclei makes the genetic manipulation of this species diffucult, so the protoplast formation is an alternative for obtaining cells with a reduced number of nuclei. Thus the experimental conditions for the production and regeneration of protoplasts inH. euphorbiae were determined by assessing three enzymatic complexes and seven osmotic stabilizers. The efficiency of formation and regeneration frequency of the protoplasts varied depending on isolates, stabilizers and enzyme mixture used. The number of nuclei estimated per protoplast was reduced to 1 to 6, depending on the stage of mycelial growth during the protoplast formation process.     

The cytokinins of cultured carrot cells

Summary Cytokinin-autotrophic strains of carrot callus contained active substances with Chromatographic mobilities on Sephadex LH-20 corresponding to 6-(4-hydroxy-3-methylbut-trans-2-enylamino)purine (Zeatin, Z), 6-(3-methylbut-2-enylamino)purine (iP) and the ribosides (9R)Z and (9R)iP. The apparent major activity was found in a fraction, with an elution volume of 242–291 ml. Hydrolysis of this fraction with HCl and -Glucosidase gave rise to Z, indicating that the major active compound is a polar conjugate of Zeatin.In all experiments the extracts were tested immediately after preparation; deep freeze storage, led to a considerable loss of activity in polar fractions. while the free base cytokinins and their ribosides showed increased activity levels.Analogous results were obtained by means of paper chromatography.     

Endogenous cytokinins in shoots of Aloe polyphylla cultured in vitro in relation to hyperhydricity, exogenous cytokinins and gelling agents

The process of hyperhydricity in tissue cultured plants of Aloe polyphylla is affected by both applied cytokinins (CKs) and the type of gelling agent used to solidify the medium. Shoots were grown on media with agar or gelrite and supplemented with different concentrations of N⁶-benzyladenine (BA) or zeatin (0, 5 and 15 μM). Endogenous CKs were measured in in vitro regenerants after an 8-weeks cycle to examine whether the hyperhydricity-inducing effect of exogenous CKs and gelling agents is associated with changes in the endogenous CK content. On media with agar a reduction in hyperhydricity occurred, while the gelrite treatment produced both normal and hyperhydric shoots (HS). The content of endogenous CKs, determined by HPLC-mass spectrometry, in the shoots grown on CK-free media comprised isopentenyladenine-, trans-zeatin- and cis-zeatin-type CKs. The application of exogenous CKs resulted in an increase in the CK content of the shoots. Following application of zeatin, dihydrozeatin-type CKs were also detected in the newly-formed shoots. Application of BA to the media led to a transition from isoprenoid CKs to aromatic CKs in the shoots. Shoots grown on gelrite media contained higher levels of endogenous CKs compared to those on agar media. Total CK content of HS was higher than that of normal shoots grown on the same medium. We suggest that the ability of exogenous CKs and gelrite to induce hyperhydricity in shoots of Aloe polyphylla is at least partially due to up-regulation of endogenous CK levels. However, hyperhydricity is a multifactor process in which different factors intervene.     

Biosynthesis of cytokinins

Cytokinins are adenine derivatives with an isoprenoid side chain and play an essential role in plant development. Plant isopentenyltransferases that catalyze the first and rate-limiting steps of cytokinin biosynthesis have recently been identified. Unlike bacterial enzymes, which catalyze the transfer of the isopentenyl moiety from dimethylallyldiphosphate (DMAPP) to the N ⁶ position of adenosine 5′-monophosphate (AMP), plant enzymes catalyze the transfer of the isopentenyl moiety from DMAPP preferentially to ATP and to ADP. The isopentenylated side chain is hydroxylated to form zeatin-type cytokinins. An alternative pathway, in which a hydroxylated side chain is directly added to the N ⁶ position of the adenine moiety, has also been suggested.     

Inhibition ofHelminthosporium oryzae andAlternaria solani byStreptomyces longisporus (Krasil'nikov) Waksman

Summary Streptomyces longisporus, isolated from field soil in association with wheat stubble, showed antagonism againstHelminthosporium oryzae andAlternaria solani in vitro as well as in soil (autoclaved). The organism or its filtrate showed formation of a distinct zone of inhibition in culture against the pathogens. Infected potato seed pieces or rice grains dipped in the culture filtrate prevented or reduced disease incidence to different degrees depending on the period of treatment. Infestation of soil (autoclaved) withS. longisporus and the pathogens in various combinations also reduced disease incidence. Among such combinations, infestation withS. longisporus followed by the pathogens proved to be the most efficient.     

Spatial and temporal changes in endogenous cytokinins in developing pea roots

Germination and seedling establishment follows a distinct pattern which is partly controlled by hormones. Roots have high levels of cytokinins. By quantifying the fluctuations in endogenous cytokinins over time, further insight may be gained into the role of cytokinins during germination and seedling establishment. Radicles were excised from sterile Pisum sativum L. seeds after 30 min and 5 h imbibition. Seedlings germinated on agar were harvested after 1, 3, 6 and 9 days. The roots were divided into the root tip, root free zone, secondary root zone and from day 6, the secondary roots. Samples were purified by various chromatographic methods and endogenous cytokinins detected by LC(+)ES-MS. Benzyladenine levels doubled after 5 h imbibition and then gradually decreased over time. Low concentrations of cis-Zeatin (cZ) type cytokinins were detected in the radicle after 30 min imbibition. After 5 h imbibition, cis-zeatin riboside-5′-monophosphate had greatly increased. The total cytokinin content of the roots increased over time with the ribotides being the predominant conjugates. From day 3 onwards, there was a gradual increase in the free bases, O-glucosides and their ribosylated forms. Mainly N ⁶ -(2-isopentenyl)adenine (iP)-type cytokinins were detected in the root tip, whereas trans-zeatin- (tZ), dihyrozeatin- (DHZ) and iP-type cytokinins were found in the secondary roots and root zone. Cytokinin biosynthesis was only detected after day 6. Biosynthesis of iP and tZ derivatives was quite rapid, whereas biosynthesis of cZ derivatives remained at a low basal level. These fluctuations in cytokinin types and concentrations suggest the cytokinins may be synthesized from various pathways in pea roots.     

Identification and quantification of the major endogenous cytokinins in pistachio seedlings

The major endogenous cytokinins, Z, ZR, DHZ, DHZR, iP and iPR in pistachio seedlings (Pistacia vera L. cv. Ohadi) were purified by HPLC and their identities confirmed using GC-MS. The aerial parts of two-year old pistachio seedlings including mature leaves, young leaves, lateral buds, debarked stems and bark were subjected to analysis. All of the above mentioned cytokinins were identified in the aerial parts except DHZ which was only present in mature leaves. Z-type cytokinins contributed almost 43% of the total cytokinins. ZR and DHZR were identified as the major ribosides and iP as the main base. The greatest concentration of ZR was detected in the bark, amounting to about 48%. DHZR and ZR constituted the major portion of the total cytokinins detected in both young and mature leaves while Z was detected as a minor cytokinin in leaves. The sharp increase of iP concentration during leaf maturation indicates that mature leaves are probably capable of de novo biosynthesis of cytokinins. The absence of DHZ (except in mature leaves) and the presence of considerable concentrations of DHZR in pistachio stems suggest that these tissues are able to metabolize DHZ to DHZR. The large amount of ZR in pistachio leaves suggests that root-derived ZR is transported into the leaves after loading into the xylem. The presence of high amounts of iP in pistachio lateral buds indicates that iP has been accumulated in these parts. The occurrence of a totally different cytokinin distribution pattern in buds, as compared with the other aerial parts, possibly results from their different metabolism.     

Involvement of cytokinins in the germination of chick-pea seeds

Eight cytokinins detected in germinated chick-pea (Cicer arietinum L. var. Castellana) seeds were first present in the embryonic axes but appeared in the cotyledons after 12h of germination. The cytokinins detected in the cotyledons originate in the embryonic axes, but no passage of these substances from the cotyledons to the axes was detected, except when the seeds were treated with red light.It is concluded that the role played by the embryonic axis in mobilizating the main reserves of the cotyledons is mainly effected through these cytokinins. Both natural and synthetic cytokinins exert an important regulatory role in the hydrolysis of reserve proteins and calcium could be involved as an intermediate.Abbreviations BA benzyladenine - cot. cotyledon - (diH)Z dihydrozeatin - (diH)ZR dihydrozeatin riboside - GZR glycosyl zeatin riboside - 2iP 277-1 - iPA 277-2 riboside - Kin kinetin - Z zeatin - ZG zeatin glucoside - ZR zeatin riboside     

Effects of cytokinins and elicitors on the production of hypericins and hyperforin metabolites in Hypericum sampsonii and Hypericum perforatum

Hypericum perforatum L. (St. John’s wort) and Hypericum sampsonii Hance are medicinal plants used in China in the treatment of viruses and other disorders. In the current study, we investigated the effects of cytokinins 6-benzylaminopurin (BA), zeatin (ZT) and thidiazuron (TDZ) on plant growth and production of hypericins (pseudohypericin and hypericin) and hyperforin. Our data suggested that culture of H. perforatum in modified MS (Murashige and Skoog) medium, with a 50% reduction in ammonium nitrate and potassium nitrate, and supplemented with BA (0.44 μM) and indolebutyric acid (IBA, 0.049 μM), resulted in increased production of hypericins. Similar results were noted with H. sampsonii with minor changes to the medium (0.46 μM ZT and 0.049 μM IBA). There were approximately 2.95-, 2.62-fold increases in H. perforatum pseudohypericin and hypericin production by TDZ (0.45 μM) induction compared to the controls. No enhancement of hypericins and hyperforin production was elicited by TDZ in H. sampsonii. The elicitor methyl jasmonate (MJA, 50 μM) and its analog, 2,3-dihydroxypropyl jasmonate (DHPJA, 50 μM), were also used in H. perforatum and H. sampsonii shoot culture to increase secondary metabolite production, eliciting an increase in the production of hypericins and hyperforin. While leaf senescence and biomass inhibition were observed in cultures induced by MJA, no such effects were observed with DHPJA.     

Inhibition of stomatal opening during uptake of carbohydrates by guard cells in isolated epidermal tissues

The uptake of glucose and other carbohydrates into the guard cells of Commelina communis L. was found to inhibit the opening of the stomata. The concentration of glucose necessary to achieve about 50% inhibition was of the same order of magnitude as the potassium concentration required for opening; the uptake systems for potassium and glucose appear to be competitive and to exhibit the same degree of affinity. It is suggested that the uptake of glucose occurs via a proton cotransport, which, depolarizing the membrane potential, slows down the electrogenic import of potassium ions. The process of stomatal closure, in contrast, appears not to be affected by carbohydrate uptake. In guard cells of Tulipa gesneriana L. and Vicia faba L., which do not possess subsidiary cells, import of glucose or other carbohydrates did not interfere with the regulation of stomatal movements.     

The carbohydrates of Phaeocystis and their degradation in the microbial food web

The ubiquity and high productivity associated with blooms of colonial Phaeocystis makes it an important contributor to the global carbon cycle. During blooms organic matter that is rich in carbohydrates is produced. We distinguish five different pools of carbohydrates produced by Phaeocystis. Like all plants and algal cells, both solitary and colonial cells produce (1) structural carbohydrates, (hetero) polysaccharides that are mainly part of the cell wall, (2) mono- and oligosaccharides, which are present as intermediates in the synthesis and catabolism of cell components, and (3) intracellular storage glucan. Colonial cells of Phaeocystis excrete (4) mucopolysaccharides, heteropolysaccharides that are the main constituent of the mucous colony matrix and (5) dissolved organic matter (DOM) rich in carbohydrates, which is mainly excreted by colonial cells. In this review the characteristics of these pools are discussed and quantitative data are summarized. During the exponential growth phase, the ratio of carbohydrate-carbon (C) to particulate organic carbon (POC) is approximately 0.1. When nutrients are limited, Phaeocystis blooms reach a stationary growth phase, during which excess energy is stored as carbohydrates. This so-called overflow metabolism increases the ratio of carbohydrate-C to POC to 0.4–0.6 during the stationary phase, leading to an increase in the C/N and C/P ratios of Phaeocystis organic matter. Overflow metabolism can be channeled towards both glucan and mucopolysaccharides. Summarizing the available data reveals that during the stationary phase of a bloom glucan contributes 0–51% to POC, whereas mucopolysaccharides contribute 5–60%. At the end of a bloom, lysis of Phaeocystis cells and deterioration of colonies leads to a massive release of DOM rich in glucan and mucopolysaccharides. Laboratory studies have revealed that this organic matter is potentially readily degradable by heterotrophic bacteria. However, observations in the field of accumulation of DOM and foam indicate that microbial degradation is hampered. The high C/N and C/P ratios of Phaeocystis organic matter may lead to nutrient limitation of microbial degradation, thereby prolonging degradation times. Over time polysaccharides tend to self-assemble into hydrogels. This may have a profound effect on carbon cycling, since hydrogels provide a vehicle to move DOM up the size spectrum to sizes subject to sedimentation. In addition, it changes the physical nature and microscale structure of the organic matter encountered by bacteria which may affect the degradation potential of the Phaeocystis organic matter.     

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 auxin and cytokinins in initiation of growth of isolated pea buds

Axillary buds from the second primary scale excised from 21-day-old pea(Pisum sativum L. cv. Vladan) plants were used as a modelsystem for studying the release of buds from apical dominance. The isolatedbudswere transferred onto basal medium with or without a supplement of growthregulators and cultivated up to 24 h in short-term and up to 4weeks in long-term experiments. In both sets of experiments endogenous IAA,cytokinins and the uptake of labelled zeatin were analysed. The development ofbuds was monitored by image analysis, estimation of their weight, and byanatomical studies. Generative meristems were found in isolated axillary budsalready in 21-day-old plants at the beginning of the experimental period. Theonset of bud growth was recorded as soon as 2 h after the budexcision by image analysis. The bud growth was accompanied by a rapid transientincrease of the endogenous IAA level within the first 2 h,followedby an increase of iPA within 24 h. The uptake of the exogenouscytokinin ([³H]Z) reached its peak between the 6 and 8hafter the release from apical dominance. The cytokinin analyses of bothshort-term and long-term bud cultures revealed the increase of free cytokininsand their glucosides, indicating de novo synthesis ofcytokinins in the buds themselves.     

Changes in carbohydrates and lipids during embryonic development ofAntheraea mylitta (Lepidoptera)

Changes in carbohydrate and lipid metabolism during embryonic development inAntheraea mylitta were studied. While carbohydrates were metabolized during early embryogenesis, lipids were catabolised at the later stages. A significant increase in both total carbohydrates and glycogen on days 5 and 6 suggested the concurrent occurrence of both gluconeogenesis and glycogenesis. As the development of the embryo proceeds, both lipids and carbohydrates were utilised, resulting in the increase in the concentration of citrate, pyruvate and lactate.     

Enzyme immunoassays of N 6-benzyladenine and N 6-(meta-hydroxybenzyl)adenine cytokinins

Enzyme-linked immunosorbent assays (ELISAs) were developed for determination of N ⁶-benzyladenosine, N ⁶-(meta-hydroxybenzyl)adenosine, and structurally related cytokinins. The use of the ELISAs allowed detection over the range of 0.05–70 pmol for N ⁶-benzyladenine and 0.01–20 pmol for the N ⁶-(meta-hydroxybenzyl)adenine cytokinins. Polyclonal antibodies used in the assays were specific for N ⁶-benzyladenine and N ⁶-(meta-hydroxybenzyl)adenine and their corresponding N ⁹-substituted derivatives. By the use of internal standardization, dilution assays, authentic [2-³H]cytokinin recovery markers, and immunohistograms, the ELISAs have been shown to be applicable for the estimation of N ⁶-benzyladenine and N ⁶-(meta-hydroxybenzyl)adenine-type cytokinins in plant tissues. For the analysis of cytokinins in the tissues of young poplar leaves and Solarium teratoma shoot culture, the extracts were fractionated by high performance liquid chromatography (HPLC) and the fractions analyzed by ELISAs. Immunohistogram ELISA analysis of fractions from different HPLC systems indicated major peaks of immunoreactivity co-chromatographing with the labeled and unlabeled standards of N ⁶-benzyladenine, N ⁶-meta-hydroxybenzyl)adenine, and their N ⁹-glycosides in these tissues.Abbreviations ELISA enzyme-linked immunosorbent assay - FW fresh weight - (mOH)[9R]BAP N ⁶-(meta-hydroxybenzyl)adenosine - HPLC high performance liquid chromatography - TBS Tris-buffered saline - TEAA triethylammonium acetate - [9R]BAP N ⁶-benzyladenosine     

Flower abscission and inflorescence carbohydrates in sensitive and non-sensitive cultivars of grapevine

The Gewurztraminer (GW) and the Pinot noir (PN) cultivars of grapevine differ in their sensitivity to environmental factors that can cause flower abscission, cv. GW being the most sensitive. In order to further define the mechanisms leading to abscission, and owing to the importance of sugars in the achievement of sexual organ ontogenesis, we attempted to correlate the chronology of flower ontogenesis with the variations of carbohydrates in the inflorescence. In the vineyard, under optimal climatic conditions, fruit set of cv. GW and cv. PN was 82% and 65%, respectively. The sugar distribution was different in their inflorescences during the entire duration of flower development. Between stages 15 and 17, flowers of GW and PN reached the crucial meiosis stage. At that time, the inflorescences of cv. GW exhibited higher concentrations of starch and sucrose, whereas those of PN presented higher levels of glucose and fructose. Despite higher starch concentrations in GW inflorescences, starch reserves were present in the ovules and anthers of PN but not in those of GW. These results suggest that the higher content of reserve and transport carbohydrates in the inflorescences of GW favour flower development and fruit set under optimal environmental conditions. Furthermore, since meiosis represents a key step of female development, the different sugar concentrations in the inflorescences of the two cultivars at stages 15 and 17 could be related to the sensitivity to flower abscission under climatic stress. In particular, the presence of starch granules in PN ovules and anthers might explain the higher resistance of this cultivar to flower abscission.     

Changes in carbohydrates and ultrastructure in xylem ray cells ofPopulus in response to chilling

Summary The ultrastructure of xylem ray cells inPopulus was studied in conjunction with their content of individual sugars and of starch. They differ considerably in structure and in carbohydrate content at the three chosen stages,i.e., of starch deposition (August), of starch maximum (November), and of starch dissolution (January). The transition from the summer to winter stage was also induced experimentally by storage of tissue at 0°C. Both in nature and after cold-storage, sucrose and its galactosides raffinose and stachyose were accumulated to a great extent, contributing up to 69.7 and 57.3% of total sugar content, respectively. They originated parallel to the breakdown of starch and to the appearance of abundant vesicular and dilated ER cisternae. Results indicating that they are the specific sites of sucrose accumulation, and/or its galactosides, are discussed. The occurrence of phytoferritin-like crystalloids in amyloplasts and of vacuolar flocculent material, which condenses into electron-dense bodies of suspectedly proteinaceous nature, is described.Dedicated to Professor Dr. Wilhelm Halbsguth on the occasion of his 75th birthday.     

Effect of cytokinins on <Emphasis Type="BoldItalic">in vitro</Emphasis> development of autotrophism and acclimatization of <Emphasis Type="Italic">Annona glabra</Emphasis> L.

The role of cytokinins in the differentiation of the photosynthetic apparatus in micropropagated plants and their effect on the plant’s ability to transition from a heterotrophic to an autotrophic condition during acclimatization was investigated. Annona glabra L. shoots were cultured on woody plant medium supplemented with sucrose and different cytokinins to evaluate leaf tissue for chloroplast development, chloroplast numbers, photosynthetic pigmentation, total photosynthetic potential, and soluble sugar content. Plants were transferred to the rooting medium in the presence or absence of sucrose and then acclimatized. Kinetin and benzyladenine (BAP) stimulated chloroplast differentiation. Inclusion of zeatin in the medium induced the formation of greater numbers of chloroplasts in the leaves, while plants cultivated in the presence of only kinetin and BAP demonstrated greater chlorophyll a and carotenoid content. The use of kinetin and BAP during in vitro culture promoted accumulation of dry matter during the acclimatization phase, especially in plants rooted under autotrophic conditions (without sucrose). Kinetin and BAP promoted development of more leaf area and greater plant survival rates in plant acclimatization on both autotrophic and heterotrophic media. The inhibitory effects of thidiazuron on the differentiation of chloroplasts, accumulation of chlorophyll a, and photosynthetic potential were examined.     

Frontiers in mycoplasma pathogenicity

Four different strains ofLactobacillus delbrueckii subsp.bulgaricus (Ss₁ and Yop₁₂) andStreptococcus salivarius subsp.thermophilus (Ss₂ and Yop₉) were isolated from two different yogurt sources in Argentina. In medium containing different carbon sources: lactose, fructose, sucrose or glucose plus fructose, the growth of a mixed culture (Yop₁₂+Ss₂) shows stimulation ofS. thermophilus and inhibition ofL. bulgaricus with respect to pure cultures. Both microorganisms in mixed culture grew less well on glucose plus galactose. However, in medium with glucose or galactose, both microorganisms were stimulated.