Aromatic Cytokinins Research Articles

Standardization of Meta-Topolin Concentration for Maximizing In vitro Proliferation in Commercial Banana Cultivars of Andhra Pradesh, India

Cytokinins are associated with cell division, cell growth, and differentiation, are the most common plant growth regulators used in micropropagation. Aromatic cytokinin, meta-topolin (mT), and its derivatives have been demonstrated as alternative cytokinins in the in vitro multiplication of various plants. Different banana cultivars differ in in vitro shoot proliferation due to the difference in endogenous growth regulator content among them. A study was conducted to figure out the effect of different concentrations of meta-topolin on in vitro shoot proliferation of banana cultivars like Karpura chakkera keli (AAB), Mortoman (AAB), and Kovvur Bontha (ABB). In vitro shoot proliferation was initiated from the first subculture (C1) itself at 1.5 ppm mT concentration in Mortoman, Kovvur Bontha, and from the second subculture (C2) at 1.0 ppm mT concentration in Kovvur Bontha. Whereas, at all other remaining concentrations, shoot proliferation of different cultivars was initiated from the third subculture (C3) only. At the end of the sixth subculture (C6), the maximum number of shoots per explant (82.70) was recorded in the Kovvur Bontha cultivar at 1.5 ppm mT concentration. Among the cultivars, the Kovvur Bontha cultivar responded better to the meta-topolin for shoot proliferation than Karpura chakkera keli (AAB) and Mortoman (AAB).

The Effect of Topophysis on the In Vitro Development of Handroanthus guayacan and on Its Metabolism of Meta-Topolin Riboside.

An important factor affecting the uniformity of in vitro cultures is the topophysical position of the original explant. We investigated this phenomenon in Handroanthus guayacan, a tropical woody tree species. Shoots from a stock culture were separated into upper, middle and basal sections and transferred to a modified MS medium containing meta-topolin-riboside and indole-butyric acid. After 8 weeks, the middle section produced the most shoots, the longest shoots and the highest number of nodes per plant. Shoots derived from the upper section were elongated, but had the shortest internodes, while those from the basal section formed the largest callus. None of the three types of explants rooted during the proliferation phase. The topophysically dependent spatial distribution of endogenous cytokinins and auxins was determined. The topophysical effect observed could not be explained solely by analyzing the endogenous isoprenoid and auxin. However, the metabolism and distribution of the aromatic cytokinin could provide an explanation. The concentration of the meta hydroxy-substituted topolins was highest in shoots derived from the middle section. Aromatic N- and O-glucosides were much more concentrated in the leaves than in the stems. In conclusion, it is recommended to consider the explant's topophysis when developing a multiplication protocol to avoid heterogeneity in an in vitro culture.

Metabolite bioprospection and expression analysis of patchoulol synthase gene in different in vitro systems of Pogostemon cablin: An important medicinal aromatic plant

Pogostemon cablin (patchouli) is one of the most prized medicinal herbs cultivated worldwide. However, with changes in the bioclimatic patterns and nematode infections in its root, the availability of quality planting material for essential oil extraction has become a challenging task in recent times. In the present research, a synergistic effort based on the identification of the optimal gene expression level of the patchoulol synthase (PS) along with fresh weight (FW), an effective strategy was formulated for the bioprospection of specialized metabolites of patchouli via plant tissue culture based in vitro approaches. The comparative expression levels of the PS gene in various tissues grown in semisolid as well as suspension cultures provided vital insights into its role in the biosynthesis of patchoulol and its associated compounds. Comparatively, cell suspension derived biomass exhibited better efficacy in terms of antioxidant activity (IC50 = 27.88 μg/ml), sun protection factor (SPF = 4.49–20.20), accumulation of both volatile and non-volatile metabolites profiled through ultra performance liquid chromatography and gas chromatography-mass spectrometry (GC-MS) along with up regulated PS gene activity (5.01 FC). Interestingly, semisolid culture media derived callus extract also exhibited potent activities establishing the novelty of the approach. The findings illustrate the utilization of aromatic cytokinin – meta-Topolin as a potent elicitor in synergy with conventional elicitors like methyl jasmonate and synthetic growth regulators such as thidiazuron. The current approach also provides vital, reproducible, sustainable and cost-effective method for the bioprospection of prized metabolites in the callus and cell suspension cultures with the goal of up-scaling to pilot scale via coordinated use of next generation bioreactor approaches.

De novo transcriptome based insights into secondary metabolite biosynthesis in Malaxis acuminata (Jeevak)-A therapeutically important orchid.

Malaxis acuminata D. Don [=Crepidium acuminatum (D. Don) Szlach.] is an endangered medicinal orchid of the Ashtvarga group of plants in Ayurveda (Indian system of traditional medicine). Using a combination of aromatic cytokinin [meta-Topolin (mT)], plant biostimulant (chitosan), auxin [indole-3-butyric acid (IBA)], and a phenolic elicitor [phloroglucinol (PG)], plants of M. acuminata were regenerated in vitro for mass multiplication. The present research reveals the first-ever transcriptome of M. acuminata. A total of 43,111 transcripts encoding 23,951 unigenes were assembled de novo from a total of 815.02 million reads obtained from leaf and pseudobulb of in vitro raised M. acuminata. Expression analysis of genes associated with β-sitosterol and eugenol biosynthesis in leaf and pseudobulb provided vital clues for differential accumulation of metabolites in M. acuminata. Ultra-performance liquid chromatography (UPLC) confirmed higher amounts of β-sitosterol and eugenol content in the leaf as compared to the pseudobulb. Differential expression of transcripts related to starch and sucrose metabolism, plant hormone signal transduction, diterpenoid biosynthesis, phenylalanine metabolism, stilbenoid, diarylheptanoid, and gingerol biosynthesis suggested the operation of differential metabolic pathways in leaf and pseudobulb. The present research provides valuable information on the biosynthesis of secondary metabolites in M. acuminata, which could be used for advanced metabolite bioprospection using cell suspension culture and bioreactor-based approaches. Data also suggested that leaf tissues rather than pseudobulb can be used as an alternate source of bioactive metabolites thereby shifting the need for harvesting the pseudobulb. This will further facilitate the conservation and sustainable utilization of this highly valued medicinal orchid.

Amelioration of Morpho-structural and Physiological Disorders in Micropropagation of Aloe vera L. by Use of an Aromatic Cytokinin 6-(3-Hydroxybenzylamino) Purine

Amelioration of Morpho-structural and Physiological Disorders in Micropropagation of Aloe vera L. by Use of an Aromatic Cytokinin 6-(3-Hydroxybenzylamino) Purine

Exogenous 6-benzylaminopurine inhibits tip growth and cytokinesis via regulating actin dynamics in the moss Physcomitrium patens.

Exogenous BAP but not 2iP disrupts actin structures and induces tip-growth retardation and cytokinesis failure in the moss Physcomitrium patens. Synthetic cytokinins have been widely used to address hormonal responses during plant development. However, exogenous cytokinins can cause a variety of cellular effects. A detailed characterization of such effects has not been well studied. Here, using Physcomitrium patens as a model, we show that the aromatic cytokinin 6-benzylaminopurine (BAP) inhibits tip growth at concentrations above 0.2µM. At higher concentrations (0.6-1µM), BAP can additionally block mitotic entry and induce cytokinesis defects and cell death. These effects are associated with altered actin dynamics and structures. By contrast, 2-isopentenyladenine (2iP) does not cause marked defects at various concentrations up to 10µM, while t-zeatin (tZ) can moderately inhibit moss growth. Our results provide mechanistic insight into the inhibitory effects of BAP on cell growth and cell division and call for attention to the use of synthetic cytokinins for bioassays.

Cytological, genetical and phytochemically stable meta-Topolin (mT) - induced mass propagation of underutilized Physalis minima L. for production of withaferin A

Abstract Physalis minima L. is commonly known as wild Gooseberry. Traditionally this plant has been used in different ethnopharmacological and modern medicinal practices. No systematic evaluation is done from an underutilized alternative resource of withaferin A content from P. minima. Withaferin A is a steroidal lactone withanolide mainly found in the plant Withania somnifera and other few members of family Solanaceae. The present investigation evaluated withaferin A content from 30 chemotypes collected from different geographical regions in India. Among 30 different chemotypes, an elite clone (17.13 ± 0.31 mg/g dry weight (DW) withaferin A) was selected for in vitro shoot multiplication. Well-organized micropropagation has been studied using five different cytokinins in Murashige and Skoog (MS) medium. Among them, aromatic cytokinin “meta-Topolin” (mT) at 1.0 mg/l triggered the highest number (83.26 ± 1.02) of multiple shoots and shoot length (4.87 ± 0.69) after 28 days of explant implantation. Maximum root induction was showed in ½ MS medium with 0.5 mg/l indole-3-butyric acid (IBA) to trigger 75.65 ± 1.03 roots and root length 4.56 ± 0.25 cm after 14 days of culture. In vitro well-rooted plantlets were excised for hardening and show; 99.02% survival rate. Genetic stability of micropropagated plants were performed with cytological and molecular marker-based selection. Simultaneously, phytochemical stability was performed with HPLC (High performance liquid chromatography) analysis. In our experiment no significant difference showed between the mother plant and micropropagated plants. Thus, our protocol can be used for large-scale commercial cultivation of elite chemotype of this medicinal plant.

The Hulks and the Deadpools of the Cytokinin Universe: A Dual Strategy for Cytokinin Production, Translocation, and Signal Transduction.

Cytokinins are plant hormones, derivatives of adenine with a side chain at the N6-position. They are involved in many physiological processes. While the metabolism of trans-zeatin and isopentenyladenine, which are considered to be highly active cytokinins, has been extensively studied, there are others with less obvious functions, such as cis-zeatin, dihydrozeatin, and aromatic cytokinins, which have been comparatively neglected. To help explain this duality, we present a novel hypothesis metaphorically comparing various cytokinin forms, enzymes of CK metabolism, and their signalling and transporter functions to the comics superheroes Hulk and Deadpool. Hulk is a powerful but short-lived creation, whilst Deadpool presents a more subtle and enduring force. With this dual framework in mind, this review compares different cytokinin metabolites, and their biosynthesis, translocation, and sensing to illustrate the different mechanisms behind the two CK strategies. This is put together and applied to a plant developmental scale and, beyond plants, to interactions with organisms of other kingdoms, to highlight where future study can benefit the understanding of plant fitness and productivity.

Direct organogenesis mediated improvised mass propagation of Pogostemon cablin: A natural reserve of pharmaceutical biomolecules

Pogostemon cablin (Blanco) Benth. or “Patchouli” is a prized medicinal herb used in various ethno-medicinal preparations and perfume industry. However due to over-collection and nematode infections in the roots of P. cablin, the wild populations are facing a steady decline and are at risk. In the present research, we report a fast clonally stable regeneration protocol for P. cablin with the utilization of meta-topolin (mT), a prized aromatic cytokinin (CK) for the first time. The highest rate of shoot proliferation i.e. 45 shoots/explant was observed after 4 weeks from leaf segments on MS media fortified with 1.0 mg/l mT. The highest rate of rooting was observed when the regenerated micro shoots were cultured in MS medium supplemented with 1.5 mg/l IBA. Direct root regeneration was tested upon with the applications of three conventional auxins i.e., Naphthalene acetic acid (NAA), Indole-3-acetic acid (IAA), Indole-3-butyric acid (IBA) using the same explants. The genetic homogeneity of the regenerated clones was ascertained, using two gene targeted molecular marker systems namely Start Codon Targeted Polymorphism (SCoT) and Inter-Retrotransposon Amplified Polymorphism (IRAP) ascertaining genetic stability amongst the regenerates and the donor plant. In precise, the present research provides vital insights into the role of aromatic CKs like mT in the high frequency regeneration of medicinally important plants like P. cablin which might serve as important baseline information for the pharma industry where supply of Quality Planting Material (QPM) is a real challenge.

The Anti-Senescence Activity of Cytokinin Arabinosides in Wheat and Arabidopsis Is Negatively Correlated with Ethylene Production.

Leaf senescence, accompanied by chlorophyll breakdown, chloroplast degradation and inhibition of photosynthesis, can be suppressed by an exogenous application of cytokinins. Two aromatic cytokinin arabinosides (6-benzylamino-9-β-d-arabinofuranosylpurines; BAPAs), 3-hydroxy- (3OHBAPA) and 3-methoxy- (3MeOBAPA) derivatives, have recently been found to possess high anti-senescence activity. Interestingly, their effect on the maintenance of chlorophyll content and maximal quantum yield of photosystem II (PSII) in detached dark-adapted leaves differed quantitatively in wheat (Triticum aestivum L. cv. Aranka) and Arabidopsis (Arabidopsis thaliana L. (Col-0)). In this work, we have found that the anti-senescence effects of 3OHBAPA and 3MeOBAPA in wheat and Arabidopsis also differ in other parameters, including the maintenance of carotenoid content and chloroplasts, rate of reduction of primary electron acceptor of PSII (QA) as well as electron transport behind QA, and partitioning of absorbed light energy in light-adapted leaves. In wheat, 3OHBAPA had a higher protective effect than 3MeOBAPA, whereas in Arabidopsis, 3MeOBAPA was the more efficient derivative. We have found that the different anti-senescent activity of 3OHBAPA and 3MeOBAPA was coupled to different ethylene production in the treated leaves: the lower the ethylene production, the higher the anti-senescence activity. 3OHBAPA and 3MeOBAPA also efficiently protected the senescing leaves of wheat and Arabidopsis against oxidative damage induced by both H2O2 and high-light treatment, which could also be connected with the low level of ethylene production.

Quantification of endogenous aromatic cytokinins in Pinus radiata embryonal masses after application of heat stress during initiation of somatic embryogenesis

Short-term exposure to high temperatures modulates the aromatic cytokinin profile of radiata pine embryonal masses when measured 8 weeks after initiation. Abstract Recent research has demonstrated that cytokinins could be involved in abiotic stress responses. However, little research has been published about the role of endogenous aromatic cytokinins during these processes. Therefore, in this study, different temperature and incubation periods (23 oC, 8 weeks; 40 oC, 4 h; 50 oC, 30 min; 60 oC, 5 min) were applied during induction of radiata pine somatic embryogenesis and the aromatic cytokinin content of the resulting embryonal masses was measured. Results indicated that temperature during induction had a significant effect on the profile of most of the aromatic cytokinins detected. All tested higher temperatures than control provoked a decline in some free bases, including N6-benzyladenine, ortho-Topolin and para-Topolin. On the other hand, the values of N6-benzyladenine nucleotides, ribosides and irreversible metabolites (9G), as well as para-Topolin ribosides, increased in embryonal masses originating from 40 oC treatment. These results suggest that despite not being traditionally considered part of the stress response machinery, aromatic cytokinins might be involved in heat-stress responses during early stages of somatic embryogenesis in radiata pine.

Micropropagation of Camptotheca Acuminata Decne (Nyssaceae) - Endangered Ornamental and Medicinal Tree

Camptotheca acuminata Decne (Nyssaceae) (happy tree, tree of life, cancer tree) is a rapidly growing deciduous and endangered tree endemic to east Tibet and southern China (Liu et al., 2002). C. acuminata is an ornamental tree with monopodial growth, beautiful leaves and shade- and cold- tolerant in their natural environment. Recently, its demand has grown rapidly due to secondary metabolites production like Camptothecin and its derivatives with anti cancer and antivirus activity. The aim of the present study was to develop an efficient protocol for in vitro micropropagation of this valuable plant. Different cultural media based on both MS (Murashige and Skoog, 1962) or DKW (Driver and Kuniyuki, 1984) formulations with different cytokinins (6-benzylaminopurine, BAP or 2-isopentenyladenine, 2iP or meta-topolin, mT) have been involved. The aromatic cytokinin meta-topolin stimulated plant growth. The optimum multiplication rate of Camptotheca shoots occurred on the DKW basal medium, supplemented with 2.5 µM meta-topolin, grown under mixed LED light. The highest number of roots per plantlets was recorded on the rooting medium with 0.3 mgl-1 NAA.

Topolins and Red Light Improve the Micropropagation Efficiency of Passion Fruit (Passiflora edulis Sims) ‘Tainung No. 1’

Passion fruit is a commercial crop of economic importance worldwide, with recent increases in demand for high-quality plants for commercial production. Plant tissue culture is widely used for the mass propagation of many commercial crops, however its application on passion fruit is challenged by the problem of low reproducibility, leaf chlorosis, and growth retardation resulted from in vitro culture. The aim of this study was to evaluate the effects of cytokinins and light quality on in vitro culture of nodal segments of passion fruit ‘Tainung No. 1’. Three aromatic cytokinins were tested in a modified MS basal medium. The bud proliferation rates of segments initiated on a media containing 1 mg·L−1 meta-topolin riboside (mTR) or benzyladenine (BA) were not significantly different at the same concentration. Buds cultured on medium supplemented with mTR grew and elongated for 4 weeks, while buds on a medium containing BA formed rosettes. After transfer to a medium without plant growth regulators (PGRs), shoots rooted spontaneously within 8 weeks. Furthermore, the effects of continuous propagation under a high proportion of red light affected the subsequent plant growth. Red LED induced an increase in the chlorophyll content (2.71 mg·g−1) compared with other light qualities (1.05–2.63 mg·g−1) and improved plantlet quality. Acclimated plants were grown in the field, and the flower morphology and fruit set were of commercial quality. Findings showed that replacing BA with mTR as the main cytokinin and using a high proportion of red light during the tissue culture induction period produced high-quality plantlets in 3 months. This system is economical and will be further developed for the commercial propagation of passion fruit vines in the future.

Embryonal Masses Induced at High Temperatures in Aleppo Pine: Cytokinin Profile and Cytological Characterization

Aleppo pine (Pinus halepensis Mill.), a native species of the Mediterranean region, has been suggested as a species that when introduced in degraded areas could facilitate the long-term colonization and expansion of late-successional species. Due to climate changes, plants need to withstand extreme environmental conditions through adaptation and changings in developmental pathways. Among other paths, plants undergo changes in developmental pathways controlled by phytohormones. At the same time, somatic embryogenesis has been widely used as a model to understand the mechanisms involved in plant response to different stresses. In this study, in order to induce a strong effect of temperature stress on plants regenerated from somatic embryos, higher temperatures (40 °C for 4 h, 50 °C for 30 min, and 60 °C for 5 min) than the control (23 °C) were applied during the induction stage of somatic embryogenesis in Pinus halepensis. A morphological characterization of the embryogenic cultures showed small differences in the number of starch grains, lipid bodies, and phenolic compounds between treatments. Results showed that high temperatures (60 °C) led to higher rates at the maturation stage of somatic embryogenesis when compared to the control (23 °C), strengthening the productivity through the increase in the number of somatic embryos obtained. Finally, analysis of endogenous concentration of cytokinins showed that different conditions applied during the initiation phase of somatic embryogenesis led to different hormonal profiles; isoprenoid cytokinins showed a clear defined pattern with the higher total hormone concentration being found in embryonal masses induced at 50 °C for 30 min, while different aromatic cytokinins presented different individual responses to the treatments applied. These differences corroborate the idea that cytokinins could be potential regulators of stress–response processes during initial steps of somatic embryogenesis.

Aromatic Cytokinin Arabinosides Promote PAMP-like Responses and Positively Regulate Leaf Longevity.

Cytokinins are plant hormones with biological functions ranging from coordination of plant growth to the regulation of biotic and abiotic stress-related responses and senescence. The components of the plant immune system can learn from past elicitations by microbial pathogens and herbivores and adapt to new threats. It is known that plants can enter the primed state of enhanced defense induced by either natural or synthetic compounds. While the involvement of cytokinins in defense priming has been documented, no comprehensive model of their action has been provided to date. Here, we report the functional characterization of two aromatic cytokinin derivatives, 6-benzylaminopurine-9-arabinosides (BAPAs), 3-methoxy-BAPA and 3-hydroxy-BAPA, that proved to be effective in delaying senescence in detached leaves while having low interactions with the cytokinin pathway. An RNA-seq profiling study on Arabidopsis leaves treated with 3-methoxy-BAPA revealed that short and extended treatments with this compound shifted the transcriptional response markedly toward defense. Both treatments revealed upregulation of genes involved in processes associated with plant innate immunity such as cell wall remodeling and upregulation of specific MAP kinases, most importantly MPK11, which is a MAPK module involved in stress-related signaling during the pathogen-associated molecular patterns (PAMPs) response. In addition, elevated levels of JA and its metabolites, jasmonate/ethylene-driven upregulation of PLANT DEFENSIN 1.2 (PDF1.2) and other defensins, and also temporarily elevated levels of reactive oxygen species marked the plant response to 3-methoxy-BAPA treatment. Synergistic interactions were observed when plants were cotreated with 3-hydroxy-BAPA and the flagellin-derived bacterial PAMP peptide (flg22), leading to the enhanced expression of the PAMP-triggered immunity (PTI) marker gene FRK1. Our data collectively show that some BAPAs can sensitively prime the PTI responses in a low micromolar range of concentrations while having no observable negative effects on the overall fitness of the plant.

Efficient Micropropagation Protocol for the Conservation of the Endangered Aloe peglerae, an Ornamental and Medicinal Species.

A number of Aloe species are facing an extremely high risk of extinction due to habitat loss and over-exploitation for medicinal and ornamental trade. The last global assessment of Aloe peglerae Schönland (in 2003) ranked its global conservation status as ‘endangered’ with a decreasing population trend. In the National Red List of South African Plants, the extremely rapid decline of this species has resulted in its conservation status being elevated from ‘endangered’ to ‘critically endangered’ based on recent or new field information. This dramatic decline necessitates the development of a simple, rapid and efficient micropropagation protocol as a conservation measure. An in vitro propagation protocol was therefore established with the regeneration of 12 shoots per shoot-tip explant within 8 weeks using Murashige and Skoog (MS) medium supplemented with 2.5 µM meta-topolin riboside (an aromatic cytokinin). The rooting of the shoots with a 100% frequency on half-strength MS medium without any plant growth resulted in additional six shoots produced per cultured shoot. The resultant plantlets were successfully acclimatized with a 100% survival frequency after 6 weeks. Overall, the developed protocol can result in the production of 3906 transplantable shoots that are ready for rooting per annum from a single shoot-tip explant. It is simple and efficient for seedling production in the ex situ cultivation and conservation of the endangered A. peglerae.

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.

New aromatic 6-substituted 2′-deoxy-9-(β)-d-ribofuranosylpurine derivatives as potential plant growth regulators

Cytokinins are naturally occurring substances that act as plant growth regulators promoting plant growth and development, including shoot initiation and branching, and also affecting apical dominance and leaf senescence. Aromatic cytokinin 6-benzylaminopurine (BAP) has been widely used in micropropagation systems and biotechnology. However, its 9-glucoside (BAP9G) accumulates in explants, causing root inhibition and growth heterogenity. To overcome BAP disadvantages, a series of ring-substituted 2′-deoxy-9-(β)-d-ribofuranosylpurine derivatives was prepared and examined in different classical cytokinin bioassays. Amaranthus, senescence and tobacco callus bioassays were employed to provide details of cytokinin activity of 2′-deoxy-9-(β)-d-ribosides compared to their respective free bases and ribosides. The prepared derivatives were also tested for their recognition by cytokinin receptors of Arabidopsis thaliana AHK3 and CRE1/AHK4. The ability of aromatic N6-substituted adenine-2′-deoxy-9-(β)-d-ribosides to promote plant growth and delay senescence was increased considerably and, in contrast to BAP, no loss of cytokinin activity at higher concentrations was observed. The presence of a 2′-deoxyribosyl moiety at the N9-position led to an increase in cytokinin activities in comparison to the free bases and ribosides. The antioxidant capacity, cytotoxicity and effect on the MHV-68 gammaherpesvirus strain were also examined.

Characterization of five CHASE-containing histidine kinase receptors from Populus × canadensis cv. Robusta sensing isoprenoid and aromatic cytokinins.

Five poplar CHASE-containing histidine kinase receptors bind cytokinins and display kinase activities. Both endogenous isoprenoid and aromatic cytokinins bind to the receptors in live cell assays. Cytokinins are phytohormones that play key roles in various developmental processes in plants. The poplar species Populus × canadensis, cv. Robusta, is the first organism found to contain aromatic cytokinins. Here, we report the functional characterization of five CHASE-containing histidine kinases from P. × canadensis: PcHK2, PcHK3a, PcHK3b, PcHK4a and PcHK4b. A qPCR analysis revealed high transcript levels of all PcHKs other than PcHK4b across multiple poplar organs. The ligand specificity was determined using a live cell Escherichia coli assay and we provide evidence based on UHPLC-MS/MS data that ribosides can be true ligands. PcHK2 exhibited higher sensitivity to iP-type cytokinins than the other receptors, while PcHK3a and PcHK3b bound these cytokinins much more weakly, because they possess two isoleucine residues that clash with the cytokinin base and destabilize its binding. All receptors display kinase activity but their activation ratios in the presence/absence of cytokinin differ significantly. PcHK4a displays over 400-fold higher kinase activity in the presence of cytokinin, suggesting involvement in strong responses to changes in cytokinin levels. trans-Zeatin was both the most abundant cytokinin in poplar and that with the highest variation in abundance, which is consistent with its strong binding to all five HKs and activation of cytokinin signaling via A-type response regulators. The aromatic cytokinins' biological significance remains unclear, their levels vary diurnally, seasonally, and annually. PcHK3 and PcHK4 display the strongest binding at pH 7.5 and 5.5, respectively, in line with their putative membrane localization in the endoplasmic reticulum and plasma membrane.

Meta-Topolin induced in vitro regeneration and metabolic profiling in Allamanda cathartica L.

An efficient micropropagation protocol has been optimized through nodal explant in Allamanda cathartica, a potential woody climber using a new aromatic cytokinin, meta-Topolin. Of the different cytokinin tested, higher number of shoots (10.5 ± 0.16) with higher shoot length (6.1 ± 0.09 cm) was observed on Murashige and Skoog (MS, 1962) medium supplemented with 3.0 μM meta-Topolin (mT). The combined effect of cytokinin-auxin (3.0 μM mT + 0.5 μM α-naphthalene acetic acid (NAA)) resulted in the production of 26.2 ± 0.32 mean shoots per explant. The inclusion of 5.0 μM ammonium nitrate (NH4NO3) to the optimized (MS + 3.0 μM mT + 0.5 μM NAA) significantly influenced the shoot procreation rate by producing 58.8 ± 0.46 mean shoots per explant in 96.80 % culture after 12 weeks of incubation. The higher rooting response in the microshoots (6 cm) was obtained on ½ MS medium containing 0.5 μM indole-3-butyric acid (IBA) which yielded a higher of 6.5 ± 0.16 mean roots with an average of 5.4 ± 0.16 cm root length in 97.80 % cultures. Among the various treated samples, higher flavonoids and quercetin content was recorded in the leaves of plantlets raised on 3.0 μM mT + 0.5 μM NAA + 5.0 μM NH4NO3, yielding 174.32 mg QE/g and 55.2 mg/g flavonoid and quercetin per gram of extract respectively. The protocol assures a speedy production of plantlets that can be used for isolation of biologically active compounds to be utilized by the pharmaceutical industries for the treatment of various diseases.