In Africa and Asia, members of the genus Brachystelma are well-known for their diverse uses, especially their medicinal and nutritional values. However, the use of many Brachystelma species as a valuable resource is generally accompanied by the concern of over-exploitation attributed to their slow growth and general small size. The aim of the current study was to establish efficient micropropagation protocols for three Brachystelma species, namely Brachystelma ngomense (endangered), Brachystelma pulchellum (vulnerable) and Brachystelma pygmaeum (least concern), as a means of ensuring their conservation and survival. This was achieved using nodal segments (~10 mm in length) as the source of explants in the presence of different concentrations of three cytokinins (CK) namely N6-benzyladenine (BA), isopentenyladenine (iP) and meta-topolin riboside (mTR), over a period of 6 weeks. The highest (25 µM) concentration of cytokinin treatments typically resulted in significantly higher shoot proliferation. However, each species differed in its response to specific CK: the optimal concentrations were 25 µM mTR, 25 µM iP and 25 µM BA for Brachystelma ngomense, Brachystelma pulchellum and Brachystelma pygmaeum, respectively. During the in vitro propagation, both Brachystelma ngomense and Brachystelma pygmaeum rooted poorly while regenerated Brachystelma pulchellum generally lacked roots regardless of the CK treatments. Following pulsing (dipping) treatment of in vitro-regenerated shoots with indole-3-butyric acid (IBA), acclimatization of all three Brachystelma species remained extremely limited due to poor rooting ex vitro. To the best of our knowledge, the current protocols provide the first successful report for these Brachystelma species. However, further research remains essential to enhance the efficiency of the devised protocol.

• Keywords
• Apocynaceae, auxins, conservation, cytokinins, meta-topolin, micropropagation, rooting,
• Publication type
• Journal Article MeSH

Cytokinins modulate a number of important developmental processes, including the last phase of leaf development, known as senescence, which is associated with chlorophyll breakdown, photosynthetic apparatus disintegration and oxidative damage. There is ample evidence that cytokinins can slow down all these senescence-accompanying changes. Here, we review relationships between the various mechanisms of action of these regulatory molecules. We highlight their connection to photosynthesis, the pivotal process that generates assimilates, however may also lead to oxidative damage. Thus, we also focus on cytokinin induction of protective responses against oxidative damage. Activation of antioxidative enzymes in senescing tissues is described as well as changes in the levels of naturally occurring antioxidative compounds, such as phenolic acids and flavonoids, in plant explants. The main goal of this review is to show how the biological activities of cytokinins may be related to their chemical structure. New links between molecular aspects of natural cytokinins and their synthetic derivatives with antisenescent properties are described. Structural motifs in cytokinin molecules that may explain why these molecules play such a significant regulatory role are outlined.

• Keywords
• antioxidant, antioxidant enzymes, antisenescent, cytokinin, derivative, genes, photosynthesis, plant defence, structure and activity relationship,
• MeSH
• Antioxidants chemistry   metabolism   MeSH
• Cytokinins chemistry   metabolism   MeSH
• Flavonoids analysis   MeSH
• Photosynthesis MeSH
• Plant Leaves chemistry   growth & development   physiology   MeSH
• Molecular Structure MeSH
• Plants chemistry   MeSH
• Plant Development MeSH
• Structure-Activity Relationship MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Antioxidants MeSH
• Cytokinins MeSH
• Flavonoids MeSH

Cytokinins (CKs) and their metabolites and derivatives are essential for cell division, plant growth regulation and development. They are typically found at minute concentrations in plant tissues containing very complicated biological matrices. Therefore, defined standards labelled with stable isotopes are required for precise metabolic profiling and quantification of CKs, as well as in vivo elucidation of CK biosynthesis in various plant species. In this work, 11 [15N]-labelled C6-purine derivatives were prepared, among them 5 aromatic (4, 5, 6, 7, 8) and 3 isoprenoid (9, 10, 11) CKs. Compared to current methods, optimized syntheses of 6-amino-9H-[15N5]-purine (adenine) and 6-chloro-9H-[15N4]-purine (6-chloropurine) were performed to achieve more effective, selective and generally easier approaches. The chemical identity and purity of prepared compounds were confirmed by physico-chemical analyses (TLC; HRMS; HPLC-MS; 1H, 13C, 15N NMR). The presented approach is applicable for the synthesis of any other desired [15N4]-labelled C6-substituted purine derivatives.

• Keywords
• 15N-labelled, cytokinin, purine, synthesis,
• Publication type
• Journal Article MeSH

Stress-induced senescence is a global agro-economic problem. Cytokinins are considered to be key plant anti-senescence hormones, but despite this practical function their use in agriculture is limited because cytokinins also inhibit root growth and development. We explored new cytokinin analogs by synthesizing a series of 1,2,3-thiadiazol-5-yl urea derivatives. The most potent compound, 1-(2-methoxy-ethyl)-3-1,2,3-thiadiazol-5-yl urea (ASES - Anti-Senescence Substance), strongly inhibited dark-induced senescence in leaves of wheat (Triticum aestivum L.) and Arabidopsis thaliana. The inhibitory effect of ASES on wheat leaf senescence was, to the best of our knowledge, the strongest of any known natural or synthetic compound. In vivo, ASES also improved the salt tolerance of young wheat plants. Interestingly, ASES did not affect root development of wheat and Arabidopsis, and molecular and classical cytokinin bioassays demonstrated that ASES exhibits very low cytokinin activity. A proteomic analysis of the ASES-treated leaves further revealed that the senescence-induced degradation of photosystem II had been very effectively blocked. Taken together, our results including data from cytokinin content analysis demonstrate that ASES delays leaf senescence by mechanism(s) different from those of cytokinins and, more effectively. No such substance has yet been described in the literature, which makes ASES an interesting tool for research of photosynthesis regulation. Its simple synthesis and high efficiency predetermine ASES to become also a potent plant stress protectant in biotechnology and agricultural industries.

• Keywords
• ASES, cytokinin, photosystem II, senescence, stress, thidiazuron, wheat,
• Publication type
• Journal Article MeSH

The CRE1/AHK4 cytokinin receptor is an important component of plants' hormone signaling systems, and compounds that can alter its activity have potential utility for studying the receptor's functions and/or developing new plant growth regulators. A high throughput method was developed for screening compounds with agonist or antagonist properties toward the CRE1/AHK4 cytokinin receptor in a single experiment using the Nanodrop II liquid handling system and 384-well plates. Potential ligands are screened directly, using a reporter system in which receptor signaling activity triggers expression of β-galactosidase in Escherichia coli. This enzyme generates a fluorescent product from a non-fluorescent substrate, allowing the agonistic/antagonistic behavior of tested compounds to be assayed in relation to that of an internal standard (here the natural ligand, trans-zeatin). The method includes a robust control procedure to determine false positive or false negative effects of the tested compounds arising from their fluorescent or fluorescent-quenching properties. The presented method enables robust, automated screening of large libraries of compounds for ability to activate or inhibit the Arabidopsis thaliana cytokinin receptor CRE1/AHK4.

• Keywords
• CRE1/AHK4 receptor, cytokinin, high throughput screening, laboratory automation, plant growth regulators,
• Publication type
• Journal Article MeSH

Dark-induced growth (skotomorphogenesis) is primarily characterized by rapid elongation of the hypocotyl. We have studied the role of abscisic acid (ABA) during the development of young tomato (Solanum lycopersicum L.) seedlings. We observed that ABA deficiency caused a reduction in hypocotyl growth at the level of cell elongation and that the growth in ABA-deficient plants could be improved by treatment with exogenous ABA, through which the plants show a concentration dependent response. In addition, ABA accumulated in dark-grown tomato seedlings that grew rapidly, whereas seedlings grown under blue light exhibited low growth rates and accumulated less ABA. We demonstrated that ABA promotes DNA endoreduplication by enhancing the expression of the genes encoding inhibitors of cyclin-dependent kinases SlKRP1 and SlKRP3 and by reducing cytokinin levels. These data were supported by the expression analysis of the genes which encode enzymes involved in ABA and CK metabolism. Our results show that ABA is essential for the process of hypocotyl elongation and that appropriate control of the endogenous level of ABA is required in order to drive the growth of etiolated seedlings.

• MeSH
• Cyclin-Dependent Kinases antagonists & inhibitors   MeSH
• Cytokinins biosynthesis   metabolism   MeSH
• Endoreduplication radiation effects   MeSH
• Homeostasis radiation effects   MeSH
• Hypocotyl cytology   growth & development   metabolism   radiation effects   MeSH
• Protein Kinase Inhibitors pharmacology   MeSH
• Germination drug effects   radiation effects   MeSH
• Abscisic Acid metabolism   MeSH
• Solanum lycopersicum cytology   growth & development   metabolism   radiation effects   MeSH
• Darkness * MeSH
• Plant Development drug effects   radiation effects   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cyclin-Dependent Kinases MeSH
• Cytokinins MeSH
• Protein Kinase Inhibitors MeSH
• Abscisic Acid MeSH