Contribution to the study of cytokinin production by phytopathogenic fungi

The excretion of cytokinins into the cultivating medium, which are produced by the phytopathogenic fungiMonilia sp. andCytospora sp. has been investigated. All the isolates of the fungi used in the experiments (Monilia fructicola, Monilia fructigena, isolate 2 and 4,Monilia laxa isolate 3 andCytospora sp. isolate CPL and C₁) have been found to produce cytokinins. The production is increased during the formation of the fructification organs. Among the isolates investigatedMonilia fructigena isolate 4 andCytospora sp. isolate CPL showed the highest production of cytokinins. After chromatographic separation, cytokinin activity was found at R_F 0.7–0.8 values by the biological test as well as by identification according to UV spectra. Application of purified cytokinins produced by the fungi evoked the formation of “green islands” on isolated barley leaves underin vitro conditions.     

Untersuchungen über rnasen in kotyledonen von nachgereiften und dormantenagrostemma githago-samen

Activities of RNasea were studied in cotyledons of dormant and afterripenedAgrostemma githago seeds. Activity of RNase increases during imbibition and germination. This increase in activity cannot be observed in variants which are not able to germinate (dormant seeds and seeds blocked by higher temperature). The development of RNase activities during germination cannot be inhibited by concentrations of cycloheximide or actinomycine D completely preventing phosphatase synthesis. These results may be indicative for the assumption that the increase of RNase during germination is caused by enzyme activation and not by enzyme synthesis. Cytokinins and a combination of cycloheximide and gibberellic acid stimulate the activity of RNase in dormant cotyledons, whereas neither cycloheximide nor gibberellic acid, applicated by themselves, show any effect. Cytokinins and gibberellic acid do not influence the activity of RNase of afterripened cotyledons, abscisic acid inhibits the increase of enzyme activity. There are characteristic changes in the pattern of RNases during germination revealed by polyacrylamide gel electrophoresis. The increase in RNase activity of dormant cotyledons caused by cytokinins is accompanied by obvious changes in the RNase pattern on polyacrylamide gel. Treating dormant cotyledons with cytokinins dormancy is partially overcome. In consequence of the application of cytokinins the differences in the electrophoretic RNase pattern between dormant and afterripened cotyledons can be nearly balanced.     

Cytopathologie von viroid-infiziertem Pflanzengewebe

Cytopathology of viroid-infected plant tissue II. Light- and electron microscopical investigations on the leaf tissue of the Chrysanthemum morifolium cultivar “Mistletoe” after infection with the chrysanthemum stunt viroid (CSV) The infection of the Chrysanthemum morifolium cultivar “Mistletoe” with the chrysanthemum stunt viroid (CSV) leads to the appearance of numerous yellowish leaf spots 2–5 mm in diameter. The cells of these chlorotic leaf areas were investigated by phase contrast- and electron microscopy and compared with the cells of the adjacent green tissue and the tissue of healthy plants. Phase contrast microscopy showed that the chlorotic tissue containes about 50 % more cells per area and that their size is reduced by 30–60 %. The parenchymatic cells of the xylem and phloem are irregular and their walls are malformed. In these cells the chloroplasts are reduced to about half in their size and number. In the electron microscope an accumulation of osmiophilic material between the thylakoid membranes of the chloroplasts of the chlorotic cells and a deterioration of the chloroplast stroma can be observed. Moreover, malformations of the cell wall and in the cell wall-associated plasmalemma-somes are found, which lead to an increase in contrast and to irregularities of their surface and internal structure. The most prominent CSV-specific cytopathic effect in cells of the vascular tissue is the extreme accumulation of microfilament bundles which were analysed in detail with the aid of a goniometer. The observed viroid-induced ultrastructural changes are compared with previously described changes caused by conventional plant viruses and the possible functional implications are discussed.     

Do cytokinins function as two‐way signals between plants and animals?

Cytokinins are plant hormones that have, among many other functions, senescence‐modulatory effects in plant tissue. This is evident not only from biochemical data, but is vividly illustrated in the “green island” phenotype in plant leaves caused by cytokinins released for example by leaf mining insects or microbial pathogens. It is beyond doubt that, in addition to their roles in plants, cytokinins also provoke physiological and developmental effects in animals. It is hypothesized that the recently much discussed modification of plant metabolism by insects and associated microbes via cytokinin signals has a counterpart in direct cytokinin signalling that interferes with the animals’ hormonal systems and impacts their population dynamics.     

Dynamics of Endogenous Cytokinins during the Growth Cycle of a Hormone-Autotrophic Genetic Tumor Line of Tobacco

The profile of endogenous cytokinins in a genetic tumor line of tobacco, namely, Nicotiana glauca (Grah.) × Nicotiana langsdorffii (Weinm.), following 1 to 10 weeks of growth on solid medium was determined by radioimmunoassay. ³H-labeled cytokinins of high specific activity were added during tissue extraction to correct for the purification losses. Following subculture (of 4-week-old tissues when their cytokinin content is high) onto fresh medium the total cytokinin content continued to be high during the first week (1470 picomoles per gram fresh weight) when the tissue fresh weight remained essentially unchanged (lag phase). The cytokinin levels then declined by about half in 2- and 3-week-old tissues (626 and 675 picomoles per gram fresh weight, respectively), a period when rapid increase in tissue fresh weight was recorded. Increments of 840% and 2780% over initial fresh weight were obtained in 2- and 3-week-old cultures, respectively. The cytokinin content then increased to initial high levels in 4-week-old tissues (1384 picomoles per gram fresh weight) after which it gradually declined with tissue age. The lowest cytokinin levels (432 picomoles per gram fresh weight) were observed in 10-week-old tissues. Maximal tissue fresh weight (4030% increase over initial fresh weight) was recorded in 5-week-old cultures after which it decreased slowly to 77.5% of the highest tissue fresh weight in 10-week-old cultures. Zeatin appeared to be the dominant endogenous cytokinin in tissues of all ages. Other cytokinins quantified were dihydrozeatin, zeatin riboside, and dihydrozeatin riboside; the values may include contributions from aglucones derived from the hydrolysis of corresponding O-glucosides, since the entire basic fraction was treated with β-glucosidase before analysis. In addition the levels of isopentenyladenine, isopentenyladenosine, and the nucleotides of zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine were also determined.     

Echinococcus granulosus: Distribution of hydatid fluid antigens in tissues of the larval stage: I. Localization of the specific antigen of hydatid fluid (antigen 5)

The indirect immunofluorescent test employing a monospecific antiserum has been used to detect the tissue localization of Echinococcus granulosus specific antigen “5.”The antigen was revealed in the inner portion of the germinal “membrane” and in the parenchyma of the protoscoleces. In these stages, it was also demonstrated fixed to the walls of some collecting ducts.It is postulated that the synthesis of the antigen “5” may occur in specialized cells of both the germinal “membrane” and the protoscoleces of the hydatid cysts.The osmoregulatory system of E. granulosus larvae seems to be involved in the transfer of the substance to the cystic cavity.     

On the Significance of Cytokinin Incorporation into RNA

The clarification of the following 2 questions was attempted: (a) are cytokinins precursors in the formation of sRNA, (b) is the observed incorporation of cytokinins into sRNA related to the action of the hormone? Although Escherichia coli contains cytokinins in its sRNA, no cytokinin auxotroph mutants of E. coli could be found and the statistical probability for the existence of such mutants is extremely low. This suggests that cytokinins are not precursors in the synthesis of sRNA. A radioactive cytokinin, 6-benzylamino-9-methyl-purine was synthesized and it was tested whether or not it is incorporated into sRNA of soybean callus tissue. Masking the 9-position of the purine inhibited the incorporation of this cytokinin into RNA while not affecting its biological activity. This is taken as an indication that the observed incorporation of cytokinins such as benzyladenine into sRNA is not related to the action of this hormone.     

A spectrophotometric cycling assay for reduced coenzyme A and its esters in small amounts of tissue.

Sensitive procedures for the assay of a few pmoles of CoASH and its esters in milligram amounts of tissue are described. The cycling method of Stadtman et al., which involves the arsenolysis of acetyl-P catalyzed by CoA and phosphotransacetylase (PTA), has been used. Selective conversion of various CoA esters to free CoA, followed by oxidation of the CoA so liberated, has enabled the specific assay of CoASH, acetyl CoA, succinyl CoA, and acetoacetyl CoA, and allows partition of the remaining CoA esters into three categories: “other PTA-reactive CoA esters,” probably mostly propionyl CoA; “PTA-unreactive CoA esters plus oxidized CoA;” and long-chain (acid-insoluble) CoA esters. Two inclusive categories are “total acid-soluble CoA” and “total CoA.” Preparation of tissue extracts is described. Rapid tissue fixation is essential for the measurement of cerebral levels of succinyl CoA, which fall 50% or more with decapitation, and of acetyl CoA, which rise 25% when the head is amputated.     

Phytohormone Involvement in the Ustilago maydis– Zea mays Pathosystem: Relationships between Abscisic Acid and Cytokinin Levels and Strain Virulence in Infected Cob Tissue

Ustilago maydis is the causative agent of common smut of corn. Early studies noted its ability to synthesize phytohormones and, more recently these growth promoting substances were confirmed as cytokinins (CKs). Cytokinins comprise a group of phytohormones commonly associated with actively dividing tissues. Lab analyses identified variation in virulence between U. maydis dikaryon and solopathogen infections of corn cob tissue. Samples from infected cob tissue were taken at sequential time points post infection and biochemical profiling was performed using high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI MS/MS). This hormone profiling revealed that there were altered levels of ABA and major CKs, with a marked reduction in CK glucosides, increases in methylthiol CKs and a particularly dramatic increase in cisZ CK forms, in U. maydis infected tissue. These changes were more pronounced in the more virulent dikaryon relative to the solopathogenic strain suggesting a role for cytokinins in moderating virulence during biotrophic infection. These findings highlight the fact that U. maydis does not simply mimic a fertilized seed but instead reprograms the host tissue. Results underscore the suitability of the Ustilago maydis– Zea mays model as a basis for investigating the control of phytohormone dynamics during biotrophic infection of plants.     

Determination by Gas Chromatography-Mass Spectrometry of [N(5)]Adenine Incorporation into Endogenous Cytokinins and the Effect of Tissue Age on Cytokinin Biosynthesis in Datura innoxia Crown Gall Tissue

In this study gas chromatographic-mass spectrometric techniques have been used to identify and quantify the metabolic incorporation of [¹⁵N₅]adenine into zeatin and its metabolites by 3-week-old Datura innoxia Mill, crown gall tissue. In a parallel study the levels of endogenous cytokinins were also determined by the stable isotope dilution technique using deuterium (²H)-labeled internal standards. Incorporation levels of the [¹⁵N₅]adenine after 8 hours of incubation, expressed as a percentage of the endogenous cytokinins, were as follows: zeatin (1.0%), zeatin riboside (1.5%), and zeatin riboside 5′-phosphate (10.2%). These results are consistent with those observed in complementary experiments using [U-¹⁴C]adenine, and support the proposal that the cytokinin biosynthesis occurs primarily at the nucleotide level. The effect of tissue age on cytokinin biosynthesis, determined by [U-¹⁴C]adenine incorporation into cytokinins by tissues at varying growth stages, indicated a steady increase with time reaching maximal synthesis at five weeks following subculture after which the level of ¹⁴C incorporation into cytokinins declined.     

Detection and identification of cytokinins produced by mycorrhizal fungi

Several fungi including six species of the genus Rhizopogon, 22 species of Hebeloma and one of Agaricus have been screened for production of cytokinins. The screening was done by culturing cytokinin-requiring soybean callus tissue alongside the fungus on a medium lacking a cytokinin supply. Growth of the soybean callus indicated production of cytokinins by the fungus. Of the fungi tested, only R. ochraceorubens A. H. Smith gave off sufficient cytokinin to be detected. Although a number of mycorrhizal species are now known to make and give off cytokinins, an even larger number apparently do not do so under the conditions of screening employed. An unidentified ectendotrophic species definitely gave off trans-zeatin, which has been crystallized, and probably trans-ribosylzeatin. Suillus punctipes (Pk.) Sing. apparently produced the same two cytokinins.     

Unabhängigkeit der bewertung zweier musterparameter von deren unterschiedlichkeitsgrad bei der Dressur

Bees were trained on a black disc (rewarded) against a smaller grey disc (unrewarded). Thus the bees had the choice of distinguishing between the two training figures by relying on the difference in size or on the difference in greyness or on both parameters. The aim of the present investigation was whether the relative weight of these parameters depended upon the differences in size and greyness presented during the training phase. 1. During the first two days of training the relative rating of these parameters changed in some, though not all individuals. In these cases the size of the discs rose in importance while the greyness was rated uniformly high (Fig. 3). Later, from the third day of training onwards the parameters were rated constantly. 2. Whenever size and greyness of the two training figures differed noticeably the bee relied on both parameters. The bee rated both parameters independently of the quantity of the differences in size and greyness presented during the training (Fig. 5). Thus the bee's analysis of the tested patterns cannot be attributed solely to the training paradigm, but seems to rely also on given (innate) “analyzers” (cf. Sutherland and Mackintosh, 1971). The type of training patterns only determines which “analyzers” influence the choice reaction. 3. The bee's “analyzer for size” had an influence on the choice reaction only if there was a noticeable size-difference between the training figures. The “analyzer for shades of grey”, however, strongly influenced the choice reactions also in those cases where the training figures showed no difference in greyness but only differences in size (Fig. 2).     

Changes in cytokinin levels of Phalaenopsis leaves at high temperature

The effect of high temperatures on cytokinin levels in Phalaenopsis hybrida leaves was investigated. Endogenous cytokinins were identified and quantified in Phalaenopsis leaves grown under high temperature conditions (30/25 °C day/night) using high performance liquid chromatography, bioassay and gas chromatography-selected ion monitoring-mass spectrometry. After 5 and 20 d of low temperature (25/20 °C day/night), zeatin, zeatin riboside and dihydrozeatin levels in the leaves were higher than that in leaves subjected to high temperature treatments. When Phalaenopsis leaves were exposed to low temperatures, about 76 % of the free cytokinins detected were of the zeatin-type. Glucoside cytokinins in the leaves increased significantly 5 d following high temperatures, and the rate of increase in glucoside cytokinins corresponded to the duration of high temperatures. At the same time, zeatin riboside and dihydrozeatin declined significantly following high temperature application. A significant accumulation of glucoside cytokinins, zeatin-9-glucoside, zeatin-O-glucoside, zeatin riboside-O-glucoside, and dihydrozeatin-O-glucoside was observed 20 d following high temperatures. These results suggest that high temperatures lead to an accumulation of glucoside cytokinins and a reduction of free base and riboside cytokinins.     

Changes in the level of endogenous cytokinins in apical buds ofChenopodium rubrum L.

CCC (2-chloroethyl)trimethylammonium chloride applied to plants ofChenopodium rubrum during floral induction led to an increase in the level of endogenous cytokinins in the apical buds. Application of gibberellic acid or indole-3-acetic acid at concentrations reversing the effect of CCC reduced the level of cytokinins. After simultaneous treatment with both CCC and one of the growth substances this reduction was less pronounced. From the comparison bf the present results, as well as of those published in previous papers it follows that in apical buds ofChenopodium rubrum there exists a mutual interaction between gibberellins and cytokinins. Under certain conditions both these groups of hormones may substitute for each other in flowering. IAA seems to affect flowering by regulating the level of both gibberellins and cytokinins.     

东北桤木叶表皮盾状腺毛的形态及其变化过程

利用电镜扫描技术,观察东北桤木叶片表面,发现其远轴面表皮上具有盾状腺毛。其由2个基细胞、4个柄细胞和20～25个头部细胞组成,随着分泌物质的积累,细胞逐渐破裂。幼叶远轴面表皮无盾状腺毛,仅有气孔分布。观察东北桤木叶片横切面,发现其为异面叶,远轴面表皮上的盾状腺毛细胞与叶脉维管组织相连。外文资料显示用于表述桤木属表皮上的盾状腺毛的名词较多,该毛状体应为“Peltate glandular hairs”。同时建议对“Glandular scales”、“Peltate gland”、“Peltate scale”、“Peltate glandular hairs”等名词进行规范统一。另外有关东北桤木叶表皮上毛状体从原表皮细胞的发生过程及其分泌物的成份,需进一步研究。     

Involvement of cis-Zeatin, Dihydrozeatin, and Aromatic Cytokinins in Germination and Seedling Establishment of Maize, Oats, and Lucerne

The aims of this study were to monitor endogenous cytokinin levels during germination and early seedling establishment in oats, maize, and lucerne to determine which cytokinin forms are involved in these processes; to quantify the transfer ribonucleic acid (tRNA)-bound cytokinins; and to measure cytokinin oxidase/dehydrogenase (CKX) activity. Cytokinins were identified using UPLC-MS/MS. The predominant free cytokinins present in the dry seeds were dihydrozeatin-type (DHZ) in lucerne and maize and cZ-type (cis-zeatin) in oats. Upon imbibition, there was a large increase in cZ-type cytokinins in lucerne although the cZ-type cytokinins remained at high levels in oats. In maize, the high concentrations of DHZ-type cytokinins decreased prior to radicle emergence. Four tRNA-bound cytokinins [cis-zeatin riboside (cZR)>N ⁶-(2-isopentenyl)adenosine (iPR), dihydrozeatin riboside (DHZR), trans-zeatin riboside (tZR)] were detected in low concentrations in all three species investigated. CKX activity was measured using an in vitro radioisotope assay. The order of substrate preference was N ⁶-(2-isopentenyl)adenine (iP)>trans-zeatin (tZ)>cZ in all three species, with activity fluctuating as germination proceeded. There was a negative correlation between CKX activity and iP concentrations and a positive correlation between CKX activity and O-glucoside levels. As O-glucosides are less resistant to CKX degradation, they may provide a readily available source of cytokinins that can be converted to physiologically active cytokinins required during germination. Aromatic cytokinins made a very small contribution to the total cytokinin pool and increased only slightly during seedling establishment, suggesting that they do not play a major role in germination.     

Cytokinin biosynthesis in crown gall tissue of Vinca rosea: Metabolism of isopentenyladenine

When isopentenyl[8-¹⁴C]adenine was incubated with crown gall tumour tissue of Vinca rosea, it was stereospecifically hydroxylated to trans-zeatin and its derivatives, which are the endogenous free cytokinins in this tissue. Adenine, adenosine and adenine nucleotides were the major degradation products.     

A guide and guard: The many faces of T-cadherin

Cadherins are a superfamily of transmembrane proteins that mediate calcium-dependent intercellular adhesion. T-cadherin (T-cad, H-cadherin or cadherin-13) is an atypical member, lacking transmembrane and cytosolic domains and possessing a glycosylphosphatidylinositol moiety that anchors T-cadherin to the plasma membrane. This article reviews current knowledge on the biomolecular characteristics of T-cadherin, its expression and function in different tissues in health and disease and its mechanisms of signal transduction. The structural characteristics of T-cadherin protein predict that it is unlikely to function as a “true” adhesion molecule in vivo. Studies from different fields suggest that it may act rather as a signalling receptor participating in recognition of the environment and regulation of cell motility, proliferation and phenotype. Cellular expression levels of T-cadherin in various tissues frequently correlate (be it negatively or positively) with the proliferative potential of the cells. Loss- and gain-of-function studies demonstrate the ability of T-cadherin to modulate cell motility and growth. Gathering evidence suggests that the “functional predestination” of T-cadherin is in control of tissue architecture through “guiding” navigation of moving structures, segregating functional tissue compartments and “guarding” integrity of functionally connected tissue layers.     

Influence des racines sur le développement végétatif ou floral des bourgeons cotylédonaires chez le Scrofularia arguta: role possible des cytokinines

Influence of roots on the vegetative or floral development of cotyledonary buds of Scrofularia arguta Sol.: A possible cytokinin role. This study shows that the presence of “nonabsorbing roots” insures a vegetative development of cotyledonary buds cultured in vitro whereas buds growing without roots produce flowers early. In the same way, roots suppress floral expression of axillary meristems of the same cotyledonary buds and induce these buds to vegetative functioning. Various trophic modifications in the culture medium are ineffective on non-rooted buds as also are gibberellin As and adenine. On the contrary, several cytokinins (kinetin, benzyladenine and zeatin) exert the same influence as roots. These results suggest that roots regulate meristematic functioning through cytokinins.