Asymmetric distribution of cytokinins determines root hydrotropism in Arabidopsis thaliana.

The phenomenon of plant root tips sensing moisture gradient in soil and growing towards higher water potential is designated as root hydrotropism, which is critical for plants to survive when water is a limited factor. Molecular mechanisms regulating such a fundamental process, however, are largely unknown. Here we report our identification that cytokinins are key signaling molecules directing root growth orientation in a hydrostimulation (moisture gradient) condition. Lower water potential side of the root tip shows more cytokinin response relative to the higher water potential side. Consequently, two cytokinin downstream type-A response regulators, ARR16 and ARR17, were found to be up-regulated at the lower water potential side, causing increased cell division in the meristem zone, which allows the root to bend towards higher water potential side. Genetic analyses indicated that various cytokinin biosynthesis and signaling mutants, including the arr16 arr17 double mutant, are significantly less responsive to hydrostimulation. Consistently, treatments with chemical inhibitors interfering with either cytokinin biosynthesis or cell division completely abolished root hydrotropic response. Asymmetrically induced expression of ARR16 or ARR17 effectively led to root bending in both wild-type and miz1, a previously known hydrotropism-defective mutant. These data demonstrate that asymmetric cytokinin distribution is a primary determinant governing root hydrotropism.

An appraisal on synthetic and pharmaceutical perspectives of pyrazolo[4,3-d]pyrimidine scaffold.

Pyrazolo[4,3-d]pyrimidine, a fused heterocycle bearing pyrazole and pyrimidine portions has gained a significant attention in the field of bioorganic and medicinal chemistry. Pyrazolo[4,3-d]pyrimidine derivatives have demonstrated numerous pharmacological activities particularly, anti-cancer, anti-infectious, phosphodiesterase inhibitors, adenosine antagonists and cytokinin antagonists etc. This review extensively unveils the synthetic and pharmacological diversity with special emphasis on structural variations around pyrazolo[4,3-d]pyrimidine scaffold. This endeavour has thus uncovered the medicinal worthiness of pyrazolo[4,3-d]pyrimidine framework. To the best of our knowledge this review is the first compilation on synthetic, medicinal and structure activity relationship (SAR) aspects of pyrazolo[4,3-d]pyrimidines since 1956.

The stay-green phenotype of wheat mutant tasg1 is associated with altered cytokinin metabolism.

KEY MESSAGE: By measuring the cytokinin content directly and testing the sensitivity to the cytokinin inhibitor lovastatin, we demonstrated that tasg1 cytokinin metabolism is different from wild-type. Our previous studies have indicated that compared with wild-type (WT) plants, a wheat stay-green mutant tasg1 exhibited delayed senescence. In this study, we found that the root development of tasg1 occurred later than that of WT. The number of lateral roots was fewer, but the lateral root length was longer in tasg1 than in WT, which resulted in a lower root to shoot ratio in tasg1 than WT. The levels of cytokinin (CK), CK activity, and expression of CK metabolic genes were measured. We found that the total CK content in the root tips and leaf of tasg1 was greater than in WT. The accumulation of mRNA of the CK synthetic gene (TaIPT) in tasg1 was higher than in WT at 9 and 11 days during seedling growth, but the expression of CK oxidase gene (TaCKX) was significantly lower in tasg1. Furthermore, the CK inhibitor lovastatin was used to inhibit CK activity. When treated with lovastatin, both the chlorophyll content and thylakoid membrane protein stability were significantly lower in tasg1 than WT, consistent with the inhibited expression of senescence-associated genes (TaSAGs) in tasg1. Lovastatin treatment also inhibited the antioxidative capability of wheat seedlings, and tasg1 was more sensitive to lovastatin than WT, as indicated by the MDA content, protein carbonylation, and antioxidant enzyme activity. The decreased antioxidative capability after lovastatin treatment may be related to the down-regulation of some antioxidase genes. These results suggest that the CK metabolism was altered in tasg1, which may play an important role in its ability to delay senescence.

Cytokinin signalling inhibitory fields provide robustness to phyllotaxis.

How biological systems generate reproducible patterns with high precision is a central question in science. The shoot apical meristem (SAM), a specialized tissue producing plant aerial organs, is a developmental system of choice to address this question. Organs are periodically initiated at the SAM at specific spatial positions and this spatiotemporal pattern defines phyllotaxis. Accumulation of the plant hormone auxin triggers organ initiation, whereas auxin depletion around organs generates inhibitory fields that are thought to be sufficient to maintain these patterns and their dynamics. Here we show that another type of hormone-based inhibitory fields, generated directly downstream of auxin by intercellular movement of the cytokinin signalling inhibitor ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6 (AHP6), is involved in regulating phyllotactic patterns. We demonstrate that AHP6-based fields establish patterns of cytokinin signalling in the meristem that contribute to the robustness of phyllotaxis by imposing a temporal sequence on organ initiation. Our findings indicate that not one but two distinct hormone-based fields may be required for achieving temporal precision during formation of reiterative structures at the SAM, thus indicating an original mechanism for providing robustness to a dynamic developmental system.

The phenylquinazoline compound S-4893 is a non-competitive cytokinin antagonist that targets Arabidopsis cytokinin receptor CRE1 and promotes root growth in Arabidopsis and rice.

We identified two phenylquinazoline compounds in a large-scale screening for cytokinin antagonists in yeast expressing the Arabidopsis cytokinin receptor cytokinin response 1/histidine kinase 4 (CRE1). After chemical modifications, we obtained compound S-4893, which non-competitively inhibited binding of the natural ligand 2-isopentenyladenine to CRE1. S-4893 antagonized cytokinin-induced activation of the Arabidopsis response regulator 5 promoter in Arabidopsis. Importantly, S-4893 had no detectable intrinsic cytokinin agonist activity in Arabidopsis or in the transformed yeast system. Cytokinin bioassay further demonstrated that S-4893 antagonized cytokinin-induced stimulation of callus formation and inhibition of root elongation. S-4893 also promoted seminal, crown and lateral root growth in rice, suggesting that S-4893 could potentially promote root growth in a variety of agronomically important plants. We believe S-4893 will be a useful tool in functional studies of cytokinin action in a wide range of plants and a lead compound for the development of useful root growth promoters in agriculture.

Signaling events leading to red-light-induced suppression of photomorphogenesis in wheat (Triticum aestivum).

Perception of red light (400 µmol photon m²/s) by the shoot bottom turned off the greening process in wheat. To understand the signaling cascade leading to this photomorphogenic response, certain signaling components were probed in seedlings grown in different light regimes. Upon analysis the gene expression of heterotrimeric Gα and Gß were severely down-regulated in seedlings grown without vermiculite and having their shoot bottom exposed to red light (R/V-) and was similar to that of dark-grown seedlings. Supplementing the red-light-grown V- seedlings with blue light resulted in up-regulation of both Gα and Gß expression, suggesting that blue light is able to modulate G protein expression. Treatment of cytokinin analog benzyladenine to cytokinin-deficient red-light-grown R/V- seedlings resulted in up-regulation of gene expression of both Gα and Gß. To probe further, modulators of signal transduction pathway--AlF3 (G protein activator), LaCl3 (Ca(2+) channel blocker), NaF (nonspecific phosphatase inhibitor), or calmodulin (CaM) antagonists trifluoperazine (TFP) and N-(6-aminohexyl)-5-chloro-1-nafthalene-sulfonamide (W-7)--were added along with Hoagland solution to the roots of 4-day-old etiolated seedlings, grown on germination paper and transferred to red light. AlF3, LaCl3, NaF failed to elicit any photomorphogenic response. However, CaM antagonists TFP and W-7 significantly reversed the red-light-induced suppression of photomorphogenesis. Phosphorylation of proteins assayed in the absence or presence of CaM antagonist TFP revealed respective up-regulation or down-regulation of phosphorylation of several plastidic proteins in R/V- seedlings. These suggest that signal transduction of red light perceived by the shoot bottom to suppress photomorphogenesis is mediated by CaM-dependent protein kinases.

Classical anticytokinins do not interact with cytokinin receptors but inhibit cyclin-dependent kinases.

Cytokinins are a class of plant hormones that regulate the cell cycle and diverse developmental and physiological processes. Several compounds have been identified that antagonize the effects of cytokinins. Based on structural similarities and competitive inhibition, it has been assumed that these anticytokinins act through a common cellular target, namely the cytokinin receptor. Here, we examined directly the possibility that various representative classical anticytokinins inhibit the Arabidopsis cytokinin receptors CRE1/AHK4 (cytokinin response 1/Arabidopsis histidine kinase 4) and AHK3 (Arabidopsis histidine kinase 3). We show that pyrrolo[2,3-d]pyrimidine and pyrazolo[4,3-d]pyrimidine anticytokinins do not act as competitors of cytokinins at the receptor level. Flow cytometry and microscopic analyses revealed that anticytokinins inhibit the cell cycle and cause disorganization of the microtubular cytoskeleton and apoptosis. This is consistent with the hypothesis that they inhibit regulatory cyclin-dependent kinase (CDK) enzymes. Biochemical studies demonstrated inhibition by selected anti-cytokinins of both Arabidopsis and human CDKs. X-ray determination of the crystal structure of a human CDK2-anticytokinin complex demonstrated that the antagonist occupies the ATP-binding site of CDK2. Finally, treatment of human cancer cell lines with anticytokinins demonstrated their ability to kill human cells with similar effectiveness as known CDK inhibitors.

Cytokinins inhibit epiphyllous plantlet development on leaves of Bryophyllum (Kalanchoë) marnierianum.

When leaves of Bryophyllum marnierianum are detached from the plant, plantlets develop from primordia located at their margins. Leaves excised with a piece of stem attached do not produce plantlets. Severing the major leaf veins overcomes the inhibitory effect of the attached stem, indicating that the control agent is transmitted through the vascular system. A possible mechanism is that an inhibitory substance, possibly a known plant hormone, transported from the stem to the leaf, suppresses plantlet development. A number of hormones were tested for their ability to inhibit plantlet primordium development in whole isolated leaves. Auxins had no effect, indicating that apical dominance is not involved. The cytokinins zeatin, kinetin, and benzylaminopurine (BAP) strongly inhibited plantlet development, suggesting that they may be the or a factor involved in maintenance of plantlet primordium dormancy when the leaf is attached to the plant. This hypothesis was strongly supported by the finding that treatment of leaves attached to stems with a cytokinin antagonist (purine riboside) released the primordia from inhibition. In contrast to whole leaves, plantlet primordium development on leaf explants incubated on Murashige Skoog medium containing 3% sucrose was strongly stimulated by cytokinins. A possible explanation of these observations is that in whole leaves the cytokinin signal is transduced into an inhibitory signal whereas in the isolated primordium cytokinin has a direct stimulatory effect. The inhibitory cytokinin pathway must be dominant as long as the leaf is attached to the plant. A model is proposed which could explain these findings. This study points to a novel role of cytokinins in the maintenance of foliar plantlet primordium dormancy.

Molecular cloning, expression, and cytokinin (6-benzylaminopurine) antagonist activity of peanut (Arachis hypogaea) lectin SL-I.

Isolation and purification of a alpha-methyl-mannoside specific lectin (SL-I) of peanut was reported earlier [Singh and Das (1994) Glycoconj J 11:282-285]. Native SL-I is a glycoprotein having approximately 31 kDa subunit molecular mass and forms dimer. The gene encoding this lectin is identified from a 6-day old peanut root cDNA library by anti-SL-I antibody and N-terminal amino acid sequence homology to the native lectin. Nucleotide sequence derived amino acid sequence of the re-SL-I shows amino acid sequence homology with the N-terminal and tryptic digests' amino acid sequence of the native SL-I (nSL-I). Presence of a putative glycosylation (QNPS) site and a hydrophobic adenine-binding (VLVSYDANS) site is also identified in SL-I. Homology modeling of the lectin suggests it to be an archetype of legume lectins. It is expressed as a approximately 30 kDa apoprotein in E. coli and has the carbohydrate specificity and secondary structure identical to its natural counterpart. The lectin SL-I inhibits cytokinin 6-benzylaminopurine (BA)-induced "delayed leaf senescence" and "cotyledon expansion". Equilibrium dialysis revealed a single high-affinity binding site for adenine (7.6 x 10(-6 )M) and BA (1.09 x 10(-5 )M) in the SL-I dimer and thus suggesting that the cytokinin antagonist effect of SL-I is mediated by the direct interaction of SL-I with BA.

Use of growth regulator of cytokinin type for enhancement and modification of herbicide activity.

The herbicidal action of Betanal Express (BPAM) on Chine jute (Abutilon theophrasti) weed was studied in the presence of a new plant growth regulator of urea type, N-phenyl-N-(1,2,4-triazol-4-yl)urea (PhenylTriazolylUrea, PTU). In the past years, Chine jute has become a major limiting factor in sugar beet production in the southern Russia due to its resistance to BPAM which is an essential herbicide widely used for sugar beet protection. When PTU was added to BPAM, the combination appeared to be more effective than the herbicide alone. The influence of phytohormone PTU was observed at very low application rate of 20-100 g/ha, thus herbicide dose in the ecosystem was reduced. The main visual signs of herbicidal action of the combination BPAM + PTU on Chine jute were inhibition of growth of overground plant and stem, leaves changes and sharp inhibition of root growth. No sugar beet injury was observed when this tank mixture was used. It was found that enhanced performance of the novel herbicide formulation is determined by increased herbicidal action of Ethofumesate, one of the active ingredients of BPAM.

Involvement of gibberellin and cytokinin in the formation of embryogenic cell clumps in carrot (Daucus carota).

Somatic embryogenesis of carrots is a typical example of the totipotency of plant cells. However, little is known about the process of change from somatic cells to embryogenic cells. To test the involvement of plant hormones in the acquisition process of embryogenic potency, we investigated the effects of plant growth regulators and their inhibitors on auxin-induced direct somatic embryogenesis of carrots. Gibberellin (GA) inhibited the early stage of embryogenic cell differentiation/development to the globular stage and uniconazole, an inhibitor of GA synthesis, promoted the secondary embryogenesis from the primary embryo. Purine riboside, an anticytokinin, inhibited direct somatic embryogenesis, and this effect was nullified by the application of cytokinin (CK). These results show that GA and CK regulate the early stage of auxin-induced somatic embryogenesis in carrots.

Inhibition of the plant cytokinin transduction pathway by bacterial histidine kinase inhibitors in Catharanthus roseus cell cultures.

We describe the isolation of two Catharanthus roseus cDNAs encoding proteins putatively involved in the final steps of a 'histidine-to-aspartate' phosphorelay in cytokinin (CK) signaling. The expression of one of these genes, CrRR1, was specifically up-regulated by CKs in C. roseus cell suspensions. We used this system as a biological model to test the activity of bacterial histidine kinase inhibitors. Our data demonstrate that these inhibitors are active on the CK transduction pathway and represent powerful chemical tools to study hormone signal transduction in plants. Moreover, these data suggest a strong conservation of functional features between prokaryotic and plant signaling pathways utilizing histidine kinases.

The polyphenol chlorogenic acid inhibits staphylococcal exotoxin-induced inflammatory cytokines and chemokines.

Proinflammatory cytokines mediate the toxic effect of staphylococcal exotoxins (SE). Chlorogenic acid, a plant polyphenol, inhibited SE-induced T-cell proliferation (by 98%) and production of interleukin 1beta, tumor necrosis factor, interleukin 6, interferon gamma, monocyte chemotactic protein I (MCP-l), macrophage inflammatory protein (MIP)-lalpha, and MIP-lbeta by human peripheral blood mononuclear cells. These data indicate that chlorogenic acid may be therapeutically useful for mitigating the pathogenic effects of SE. Naturally occurring polyphenolic compounds such as chlorogenic acid may serve as a potent anti-inflammatory agent alternative to conventional chemotherapeutics.

Inhibition of cytokine production and endothelial expression of adhesion antigens by 5'-methylthioadenosine.

We investigated the activity of endogenous nucleoside 5'-deoxy-5'-methylthioadenosine (MTA) on both the production of inflammatory cytokines and the cytokine-dependent endothelial expression of adhesion molecules. The compound inhibited the production of tumor necrosis factor (but not interleukin-1) in lipopolysaccharide-activated macrophages. In addition, MTA selectively inhibited the expression of intercellular adhesion molecule-1 in endothelial cells activated with interleukin-1. This effect was paralleled by a reduction in endothelial adhesiveness for polymorphonuclear leukocytes. These data suggest that MTA might have anti-inflammatory activity.

Hormonal induction and antihormonal inhibition of tracheary element differentiation in Zinnia cell cultures.

Mechanically isolated mesophyll cells of Zinnia elegans L. cv Envy differentiate to tracheary elements when cultured in inductive medium containing sufficient auxin and cytokinin. Tracheary element differentiation was induced by the three auxins (alpha-naphthaleneacetic acid, indole-3-acetic acid, and 2,4-dichlorophenoxyacetic acid) and four cytokinins (6-benzyladenine, kinetin, 2-isopentenyladenine and zeatin) tested. Tracheary element formation is inhibited or delayed if the inductive medium is supplemented with an anticytokinin, antiauxin, or inhibitor of auxin transport.

4-Coumarate:coenzyme A ligase and isoperoxidase expression in Zinnia mesophyll cells induced to differentiate into tracheary elements.

When cultured in inductive medium containing adequate auxin and cytokinin, isolated mesophyll cells of Zinnia elegans L. cv Envy differentiate into tracheary elements with lignified secondary wall thickenings. Differentiation does not occur when cells are cultured in control medium, which has reduced levels of auxin and/or cytokinin. The activities of two enzymes involved in lignin synthesis, 4-coumarate:coenzyme A ligase and peroxidase, were examined. An induction-specific cationic isoperoxidase, visualized by low pH polyacrylamide gel electrophoresis, is detectable in soluble and wall fractions of cultured Zinnia cells long before tracheary elements visibly differentiate and is thus an early marker of differentiation. Compounds (such as antiauxins, anticytokinins, and tunicamycin) that inhibit or delay differentiation alter the expression of this isoperoxidase. 4-Coumarate:coenzyme A ligase activity increases dramatically only as cells differentiate. Together, these results suggest that the onset of lignification in differentiating Zinnia cells might be controlled by the availability of precursors synthesized by way of 4-coumarate:coenzyme A ligase. These precursors would then be polymerized into lignin in the cell wall by the induction-specific isoperoxidase.

Fluorescent anticytokinins as a probe for binding. Isolation of cytokinin-binding proteins from the soluble fraction and identification of a cytokinin-binding site on ribosomes of tobacco callus cells.

4-Substituted 2-methylthiopyrido[2,3-d]pyrimidines, a series of recently developed anticytokinins, have been found to fluoresce strongly in water and to be useful as probes for binding studies. The binding activity of the soluble proteins and particulate fraction of tobacco callus cells to the biologically most active member of the family, 4-n-butylamino-2-methylthiopyrido[2,3-d]pyrimidine (BAMPP), was studied fluorimetrically. We found that the binding activity is better monitored in terms of saturable binding rather than in terms of the amount of bound ligand, a conventional method used in isolation studies of hormone receptor proteins. Using this technique we isolated two kinds of high-affinity cytokinin-binding proteins from the soluble fraction and identified a high-affinity binding site on ribosomes.