Natural Cytokinins Research Articles

Anti-Proliferative Activity of Ethylenediurea Derivatives with Alkyl and Oxygen-Containing Groups as Substituents

Background/Objectives: Natural cytokinins are a promising group of anti-tumor agents. In this work, we hypothesized that modification of the ethylenediurea moiety with alkyl and oxygen-containing groups could be a way to enhance the anti-proliferative properties of the molecule. Methods: Ten new analogs of ethylenediurea with these substitutions were tested for anti-proliferative activity in the human cancer cell lines MDA-MB-231 (breast cancer), A-375 (melanoma), and U-87 MG (glioblastoma) during 72 h of incubation using resazurin test and evaluated the substances receptor using molecular docking. Results: The compound with the carbamate link and ethylene substituent on the phenyl ring inhibited proliferation in these models by 70–90% without cytotoxic effects. The compound did not affect the viability of the immortalized fibroblast cell line Bj-5ta. The compound was also able to enhance the action of doxorubicin and temozolomide by about 20%. According to the molecular modeling data, the probable receptor target for the synthesized compound was the A2AR adenosine receptor. Conclusions: The results obtained on the ethylenediurea analogs with ethyl substituent in the aromatic ring are promising for the development of novel anticancer therapeutics.

Organic Supplementation of Vaccinium corymbosum Micropropagation Media.

The best Vaccinium corymbosum plant growth under in vitro conditions can be achieved by using the right composition and pH of the medium. For the initial phase of in vitro culture, a combination of cytokinins-mostly zeatin-can usually be used. Organic supplementation of the medium enables the use of a replacement for the expensive natural cytokinin used in micropropagation of highbush blueberry. This chapter describes the experiments with silicon Hydroplus™ Actisil (Si), coconut water (CW), and different pH (5.0; 5.5, and 6.0) as a stress factor. The addition of 200mgdm-3 silicon solution and 15% coconut water strongly stimulated highbush blueberry plant growth in vitro. Moreover, silicon solution benefits the negative effects of higher pH of the medium used for micropropagation of V. corymbosum. Maximum vegetative development of blueberry explants was obtained at pH5.

The Cytokinins BAP and 2-iP Modulate Different Molecular Mechanisms on Shoot Proliferation and Root Development in Lemongrass (Cymbopogon citratus).

The known activities of cytokinins (CKs) are promoting shoot multiplication, root growth inhibition, and delaying senescence. 6-Benzylaminopurine (BAP) has been the most effective CK to induce shoot proliferation in cereal and grasses. Previously, we reported that in lemongrass (Cymbopogon citratus) micropropagation, BAP 10 µM induces high shoot proliferation, while the natural CK 6-(γ,γ-Dimethylallylamino)purine (2-iP) 10 µM shows less pronounced effects and developed rooting. To understand the molecular mechanisms involved, we perform a protein-protein interaction (PPI) network based on the genes of Brachypodium distachyon involved in shoot proliferation/repression, cell cycle, stem cell maintenance, auxin response factors, and CK signaling to analyze the molecular mechanisms in BAP versus 2-iP plants. A different pattern of gene expression was observed between BAP- versus 2-iP-treated plants. In shoots derived from BAP, we found upregulated genes that have already been demonstrated to be involved in de novo shoot proliferation development in several plant species; CK receptors (AHK3, ARR1), stem cell maintenance (STM, REV and CLV3), cell cycle regulation (CDKA-CYCD3 complex), as well as the auxin response factor (ARF5) and CK metabolism (CKX1). In contrast, in the 2-iP culture medium, there was an upregulation of genes involved in shoot repression (BRC1, MAX3), ARR4, a type A-response regulator (RR), and auxin metabolism (SHY2).

Obtaining and Studying the Properties of Chitosan Films Containing Natural Phytohormones Cytokinins

A promising carrier for the development of polymer systems with controlled release of biologically active compounds is the aminopolysaccharide chitosan. In the present work, we studied the possibility of using chitosan films as a matrix for the N6-benzyladenine (BA), which is the natural cytokinin widely used in tissue culture. The aim of this work was to develop biopolymer carriers containing phytohormones cytokinins that provide its controlled release. As a result of the work, a number of biopolymer carriers containing BA were obtained, and the kinetics of moisture absorption of the resulting complexes and the kinetics of their release of cytokinins were studied. It has been shown that the use of a polymer carrier based on chitosan is a convenient matrix for achieving a prolonged biological effect from cytokinins. The obtained results will make it possible to purposefully design materials with an optimal delivery rate of cytokinins for a biological object.

Topolin cytokinins enhanced shoot proliferation, reduced hyperhydricity and altered cytokinin metabolism in Pistacia vera L. seedling explants

The effect of 10 µM meta-topolin (mT) and meta-topolin riboside (mTR) on in vitro proliferation and anomalies of Pistacia vera L. were evaluated and compared to that of 6-benzylaminopurine (BA). The highest proliferation rate (15.6) was recorded in the mT-medium, with a value 6 times higher than in BA-medium. Moreover, the lowest percentage of hyperhydric usable shoots (58,9%) and callus weight (46,9%) were found in mTR-treated shoots. Shoot tip as well as leaf necrosis were not influenced by cytokinin (CK) type. Image analysis was used to evaluate photosynthetic efficiency as well as anthocyanin index. Photosynthesis was more efficient with BA and mTR but the higher anthocyanin accumulation in BA-treated shoots suggests more stress. Endogenous CKs and their metabolites were determined in seedlings and, for the first time, the metabolism of exogenous BA, mT and mTR was studied in pistachio. The stimulating effect on cis-zeatin and its riboside and the appearance of BA and traces of ortho-topolin and para-topolin as natural CKs are discussed. The quantitative and qualitative CK metabolite analyses provides some initial clues as to why topolin would be superior to BA in terms of proliferation rate and avoiding hyperhydricity and allowed a better understanding of the effect of exogenous administration of CK.

Effect of Paisen, Populuseuphratica Bud Extract and Copper Sulfate on Some Traits of Vegetative Growth and Yield of Aswad Diyala Cultivars

This study was conducted in a private orchard in Al-Kifl District/Babylon province for the season 2020, 54 fig trees of the Aswad Diyala cultivar, aged 8 years, were selected, homogeneous in size and growing strength as much as possible and planted on dimensions (5 x 5 m). Trees are irrigated as a tourist and fertilized with nitrogen and compound fertilizers in two batches in the third and fifth months annually at an average of 1 kg/tree-1, as well as fertilized with decomposed organic fertilizer every two years depending on a source for fertilizer recommendations. The study included 18 treatments, and each treatment was replicated three times (each tree was considered an experimental unit). The randomized complete block design (R.C.B.D) ‘Research design: The experiment is implemented by following the Randomized Complete Block Design (R.C.B.D) as a factorial experiment (3 x 3 x 2) of Paisen were (0, 100, 200) mg/L-1 (Paisen is a natural cytokinin extracted from the roots of grape vines) and Populuseuphratica bud extract (0, 20, 30) g/L-1 and copper sulfate (0, 2.5) g/L-1 spraying on the vegetative growth in three periods. And the highest results obtained were for) Paisen 200 mg/L-1 + extract 30 g/L-1 + copper sulfate 2.5 g/L-1 ( (yield quantity - fruit weight - fruit size - chlorophyll content of leaves and leaf area) and were recorded (27.12 kg – 38.05 g – 33.97cm3 – 115.93 mg/kg fresh weight – 174.19 cm2 ) respectively) when using the highest concentrations of each of the study factors combined compared to the lowest results when the control treatment and reached ( 21.03 kg – 30.68 g – 25.07 cm3 – 111.77 mg/kg fresh weight – 152.26 cm2) respectively.

The adjuvant activity of two urea derivatives on cytokinins: an example of serendipitous dual effect

The aim of this study was to investigate the action spectrum of two urea derivatives, the 1,3-di(benzo[d]oxazol-5-yl)urea (5-BDPU) and the 1,3-di(benzo[d]oxazol-6-yl)urea (6-BDPU). In order to evaluate a possible adjuvant activity on cytokinins the compounds alone or in the simultaneous presence of different cytokinins were assayed either on in vitro typical cytokinin-related bioassays, or on in planta interaction with cytokinin signal transduction pathway. The compounds ability to activate the cytokinin receptor CRE1/AHK4 was studied either by a heterologous bacterial assay or by a competitive binding assay and docking simulations were performed with the crystal structure of the same receptor. Then, owing to their chemical structure which resembles that of urea-type cytokinins, the ability of 5- and 6-BDPU to inhibit the activity of cytokinin oxidase/dehydrogenase of Zea mays (ZmCKX1) was investigated and docking simulations were performed as well. Accordingly to the experimental results, we speculate that BDPUs could show a dual activity: the blocking of the conformational re-adaption of CRE1/AHK4 receptor maintaining the cytokinin inside its binding pocket, thus possibly enhancing its kinase action; the inhibition of cytokinin oxidase/dehydrogenase activity thus possibly preventing its cleavage of natural cytokinins with isoprenoid side chain.Graphic abstract

Cytokinin Plant Hormones Have Neuroprotective Activity in In Vitro Models of Parkinson's Disease.

Cytokinins are adenine-based phytohormones that regulate key processes in plants, such as cell division and differentiation, root and shoot growth, apical dominance, branching, and seed germination. In preliminary studies, they have also shown protective activities against human neurodegenerative diseases. To extend knowledge of the protection (protective activity) they offer, we investigated activities of natural cytokinins against salsolinol (SAL)-induced toxicity (a Parkinson’s disease model) and glutamate (Glu)-induced death of neuron-like dopaminergic SH-SY5Y cells. We found that kinetin-3-glucoside, cis-zeatin riboside, and N6-isopentenyladenosine were active in the SAL-induced PD model. In addition, trans-, cis-zeatin, and kinetin along with the iron chelator deferoxamine (DFO) and the necroptosis inhibitor necrostatin 1 (NEC-1) significantly reduced cell death rates in the Glu-induced model. Lactate dehydrogenase assays revealed that the cytokinins provided lower neuroprotective activity than DFO and NEC-1. Moreover, they reduced apoptotic caspase-3/7 activities less strongly than DFO. However, the cytokinins had very similar effects to DFO and NEC-1 on superoxide radical production. Overall, they showed protective activity in the SAL-induced model of parkinsonian neuronal cell death and Glu-induced model of oxidative damage mainly by reduction of oxidative stress.

PENGARUH EKSTRAK Sargassum cristaefolium PADA MULTIPLIKASI Dendrobium antennatum Rchb.f SECARA IN VITRO

The purpose of this study was to determine the potential and concentration of Sargassum cristaefolium extract as a natural cytokinin in tissue culture media of Dendrobium antennatum Rchb.f. This study is experimental with a completely randomized design, using several extract concentrations compared with the positive control (BAP 1.5 ppm) and negative control (MS0 media). Extract concentrations used 5 ppm, 10 ppm, 15 ppm, 20 ppm, and 25 ppm. Based on analysis of variance (ANOVA), the effect of Sargassum cristaefolium extract on the growth media significant on all parameters. Sargassum cristaefolium extracts caused different responses at certain levels of concentration. Extract concentration of 10 ppm was able to initiate the highest number of shoots and leaves compared to other extract concentrations, where as the concentration 20 ppm was able to accelerate and increase root growth.

Cytokinin fluoroprobe reveals multiple sites of cytokinin perception at plasma membrane and endoplasmic reticulum

Plant hormone cytokinins are perceived by a subfamily of sensor histidine kinases (HKs), which via a two-component phosphorelay cascade activate transcriptional responses in the nucleus. Subcellular localization of the receptors proposed the endoplasmic reticulum (ER) membrane as a principal cytokinin perception site, while study of cytokinin transport pointed to the plasma membrane (PM)-mediated cytokinin signalling. Here, by detailed monitoring of subcellular localizations of the fluorescently labelled natural cytokinin probe and the receptor ARABIDOPSIS HISTIDINE KINASE 4 (CRE1/AHK4) fused to GFP reporter, we show that pools of the ER-located cytokinin receptors can enter the secretory pathway and reach the PM in cells of the root apical meristem, and the cell plate of dividing meristematic cells. Brefeldin A (BFA) experiments revealed vesicular recycling of the receptor and its accumulation in BFA compartments. We provide a revised view on cytokinin signalling and the possibility of multiple sites of perception at PM and ER.

Distinct Peculiarities of In Planta Synthesis of Isoprenoid and Aromatic Cytokinins.

The biosynthesis of aromatic cytokinins in planta, unlike isoprenoid cytokinins, is still unknown. To compare the final steps of biosynthesis pathways of aromatic and isoprenoid cytokinins, we synthesized a series of nucleoside derivatives of natural cytokinins starting from acyl-protected ribofuranosyl-, 2′-deoxyribofuranosyl- and 5′-deoxyribofuranosyladenine derivatives using stereoselective alkylation with further deblocking. Their cytokinin activity was determined in two bioassays based on model plants Arabidopsis thaliana and Amaranthus caudatus. Unlike active cytokinins-bases, cytokinin nucleosides lack the hormonal activity until the ribose moiety is removed. According to our experiments, ribo-, 2′-deoxyribo- and 5′-deoxyribo-derivatives of isoprenoid cytokinin N6-isopentenyladenine turned in planta into active cytokinins with clear hormonal activity. As for aromatic cytokinins, both 2′-deoxyribo- and 5′-deoxyribo-derivatives did not exhibit analogous activity in Arabidopsis. The 5′-deoxyribo-derivatives cannot be phosphorylated enzymatically in vivo; therefore, they cannot be “activated” by the direct LOG-mediated cleavage, largely occurring with cytokinin ribonucleotides in plant cells. The contrasting effects exerted by deoxyribonucleosides of isoprenoid (true hormonal activity) and aromatic (almost no activity) cytokinins indicate a significant difference in the biosynthesis of these compounds.

Comparative analysis of the biosynthesis of isoprenoid and aromatic cytokinins

To compare the biosynthesis pathways of aromatic and isoprenoid cytokinins, a series of nucleoside derivatives of natural cytokinins was synthesized and their cytokinin activity was determined in a test system based on the model plant Arabidopsis thaliana. Cytokinin nucleosides are known to lack the hormonal activity until cleaving the ribose moiety at the position 9. Our experiments have shown that both ribo- and 5-deoxyribo derivatives of N 6-isopentenyladenine were able to turn into active cytokinins in planta exhibiting cytokinin activity. By contrast, 5-deoxy nucleosides of aromatic cytokinins did not show similar activity. Since 5-deoxy nucleosides cannot phosphorylate in vivo, the direct pathway of active cytokinin formation by cleavage of nucleotides is blocked here. The detected activity in 5-deoxy nucleosides of isoprenoid cytokinins and the lack of the activity in 5-deoxy nucleosides of aromatic cytokinins indicates a difference in the biosynthesis of these compounds.

Adsorption of Trans-Zeatin on Laser-Ablated Gold Nanoparticles for Transport into Plant Cells and Growth Stimulation

Gold nanoparticles can serve as nanovectors for trans-zeatin, a natural cytokinin used in plant culture to stimulate growth and bud formation. Here, we have used Raman scattering, X-ray photoelectr...

Role of Cytokinins in Senescence, Antioxidant Defence and Photosynthesis.

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.

Synthesis of Cytokinins via Enzymatic Arsenolysis of Purine Nucleosides.

This unit describes an effective method for the preparation of natural cytokinins and their synthetic derivatives based on enzymatic cleavage of the N-glycosidic bond of N6 -substituted adenosine or O6 -substituted inosine derivatives in the presence of purine nucleoside phosphorylase (PNP) and Na2 HAsO4 . The arsenolysis reaction is irreversible due to the hydrolysis of the resulting α-D-ribose-1-arsenate. As a result, the desired products are formed in near-quantitative yields, as indicated by high-performance liquid chromatography (HPLC) analysis, and can easily be isolated. In the strategy used here, the ribose residue acts as a protective group. © 2018 by John Wiley & Sons, Inc.

Profiles of Little-Known Medicinal Polypores: Funalia trogii (Agaricomycetes).

The purpose of this study was to comprehensively characterize little-known polypores that have recently been found to possess anticancer activity and thus can be used in targeted cancer therapy. Funalia trogii is a polypore with bipolar distribution and has been found by harvesters working in taiga forests, broadleaf forests, and forest-steppes of the Holarctic, and in semiarid temperate forests of the Southern Hemisphere. Substances such as gibberellic acid, abscisic acid, indole-3-acetic acid, and natural cytokinin were determined in culture media of F. trogii. Also, laccases and peroxidases of spare action have been reported in F. trogii culture media. All of the aforementioned substances can be used in targeted cancer therapy, but further investigation of F. trogii is recommended; more details of its health benefits could expand its use in mycotherapy.

Natural plant hormones cytokinins increase stress resistance and longevity of Caenorhabditis elegans

Cytokinins are phytohormones that are involved in many processes in plants, including growth, differentiation and leaf senescence. However, they also have various activities in animals. For example, kinetin and trans-zeatin can reduce levels of several aging markers in human fibroblasts. Kinetin can also protect mice against oxidative and glyoxidative stress, and prolong fruit flies' lifespan. Additionally, several cytokinins are currently used in cosmetics. To extend knowledge of the breadth of cytokinins' activities, we examined effects of natural cytokinin bases on the model nematode Caenorhabditis elegans. We found that kinetin, para-topolin and meta-topolin prolonged the lifespan of C. elegans. Kinetin also protected the organism against oxidative and heat stress. Furthermore, our results suggest that presence of reactive oxygen species, but not DAF-16 (the main effector of the insulin/insulin-like growth factor signaling pathway), is required for the beneficial effects of kinetin. Ultra-high performance liquid chromatography-tandem mass spectrometric analysis showed that kinetin is unlikely to occur naturally in C. elegans, but the worm efficiently absorbs and metabolizes it into kinetin riboside and kinetin riboside-5'-monophosphate.

The effect of benzylaminopurine on fatty-acid composition of membrane lipids in shoots of Karelian birch in vitro

The effect of the synthetic analogue of natural cytokinin 6-benzylaminopurine (BAP) on the fatty-acid composition of membrane lipids in shoots of Karelian (Curly) birch Betula pendula Roth var. carelica (Mercklin) Hamet-Ahti under in vitro conditions was studied. It was found that the addition of BAP (in a concentration of 0.25, 0.5, 1.0, or 2 mg/L) to the nutrient medium leads to an increase in the proportion of saturated fatty acids in phospholipids, whereas in the control variant unsaturated fatty acids predominated in phospholipids. In glycolipids, on the contrary, BAP caused an increase in the synthesis of unsaturated fatty acids (predominantly, linolenic), but the degree of its effect depended on the concentration of the hormone. In particular, when the maximum amount of the studied concentration (2.0 mg/L) was used, the use of BAP was shown to decrease the proportion of linolenic acid. It is suggested that the revealed changes in the composition of glycolipids are indicators of the stimulating effect of cytokinin (up to a certain concentration) on the structure and function of chloroplasts. Due to the photomixotrophy of shoot cultures, chloroplasts are an important factor determining not only their photosynthetic activity, but also cell proliferation and the direction of in vitro morphogenesis.

Fluorination of Naturally Occurring N⁶-Benzyladenosine Remarkably Increased Its Antiviral Activity and Selectivity.

Recently, we demonstrated that the natural cytokinin nucleosides N6-isopentenyladenosine (iPR) and N6-benzyladenosine (BAPR) exert a potent and selective antiviral effect on the replication of human enterovirus 71. In order to further characterize the antiviral profile of this class of compounds, we generated a series of fluorinated derivatives of BAPR and evaluated their activity on the replication of human enterovirus 71 in a cytopathic effect (CPE) reduction assay. The monofluorination of the BAPR-phenyl group changed the selectivity index (SI) slightly because of the concomitant high cell toxicity. Interestingly, the incorporation of a second fluorine atom resulted in a dramatic improvement of selectivity. Moreover, N6-trifluoromethylbenzyladenosine derivatives (9–11) exhibited also a very interesting profile, with low cytotoxicity observed. In particular, the analogue N6-(3-trifluoromethylbenzyl)-adenosine (10) with a four-fold gain in potency as compared to BAPR and the best SI in the class represents a promising candidate for further development.

Chemical proteomic analysis of 6-benzylaminopurine molecular partners in wheat grains.

An affinity-based chemical proteomic technique enabled direct identification of BAP-interacting proteins in wheat, including the well-known cytokinin-binder, cytokinin-binding protein 1. In this work, we show the development of a chemical proteomic technique for the identification of proteins binding to natural aromatic cytokinins (CKs). 6-benzylaminopurine (BAP) and documented CK-binder, wheat germ-allocated cytokinin-binding protein 1 (CBP-1), were suggested as an ideal proof-of concept affinity pair. Therefore, wheat grains were chosen as a model plant material. The BAP affinity beads were prepared by the immobilization of synthesized BAP-derived ligand to a commercial, pre-activated resin and used to isolate target proteins. The proteomic analysis of complex plant extracts is often complicated by the presence of highly abundant background proteins; in this case, the omnipresent alpha-amylase inhibitors (AAIs). To cope with this problem, we included SDS-PAGE, in-gel trypsin digestion and fraction pooling prior to shotgun analysis, which brought about an obvious drop in the signals belonging to the obstructing proteins. This was accompanied by a sharp increase in the number of identified BAP targets in comparison to a conventional in-solution digestion approach. To distinguish specific CK-binding proteins from those having a general affinity for nucleotide-like compounds, competitive pull-downs with natural nucleotides and free BAP were included in every affinity experiment. By this approach, we were able to identify a group of BAP-interacting proteins, which were subsequently found to be related to biological processes affected by CKs. Moreover, the selected affinity enrichment strategy was verified by the detection of the aforementioned CK-interacting protein, CBP-1. We propose that the developed method represents a promising tool for appealing research of as yet unknown CK molecular partners in plants.