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In this study, 4-vinylphenylboronic acid, ionic liquid, and dipentaerythritolhexakis(3-mercaptopropionate) were used as functional monomers to prepare monolithic column in fused-silica capillaries by thermally induced sulfhydryl-alkenyl click polymerization. Based on the properties of 4-vinylphenylboronic acid and ionic liquid, the monolithic capillary column (VPBA-SH-IL) exhibited excellent permeability and chemical stability with the ability to efficiently enrich trace amounts of cytokinins from complex substrates via B←N coordination, π-π interactions, cation-π interactions, S-π interactions and hydrophobic interactions. Under the optimal adsorption conditions, the adsorption efficiency of VPBA-SH-IL for six cytokinins could reach more than 95 %, and the adsorption capacities ranged from 0.18 to 0.99 mg·m -1 . A reliable and sensitive method for the determination of six cytokinins in organs of tomato (root, stem, leaf and fruit) was established by using the monolithic capillary column as in-tube solid-phase microextraction coupled with UHPLC-MS/MS. The developed method showed a wide linear range (0.010-10.0 μg·L -1 ) and low limits of detection (1.6-3.3 ng·L -1 ). The recoveries of the six cytokinins in spiked samples ranged from 80.6 % to 108 % with the relative standard deviations of 2.4-8.7 % (n = 5). The VPBA-SH-IL shows good potential for extraction and analysis of trace cytokinins in complex matrices such as plants, which is of great significance in exploring the mechanism of plant growth and development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The study investigated the adaptability of Moringa oleifera to saline conditions, focusing on its hormesis behavior. It also examined how various plant growth regulators affected growth, physiological parameters, and bioactive compounds of moringa. In the first phase, different NaCl stress levels (0, 50, 100, 150, 200, and 250 mM) were applied. Notably, significant stimulation was observed at 100 mM stress for growth, total phenolics, total flavonoids and total chlorophyll content while 150 mM stress had a marked inhibitory effect, with survival decreasing at 200 and 250 mM NaCl levels. A 38% reduction in root attributes and shoot length, along with a 55% decrease in leaf score, was observed at 150 mM stress. Total phenolics showed a positive correlation with growth attributes. In the second phase, moringa plants grown under 50, 100, and 150 mM NaCl stress were treated with various plant growth regulators, including cytokinin (50 mg L -1 ), thiourea (5 mM), bezyl amino purine (BAP @50 mg L -1 ), salicylic acid (50 mg L -1 ), hydrogen peroxide (H 2 O 2 @120 μM), or ascorbic acid (50 mg L -1 ) to mitigate adverse effects of salinity. Cytokinin, BAP, and salicylic acid applications improved salinity tolerance, enhancing enzymatic, and non-enzymatic antioxidants, and the abundance of kaempferol, quercetin, hydroxybenzoic, and hydroxycinnamic acids. Pearson correlation and principal component analysis manifested relationships among growth parameters, antioxidant activities, flavonoids, and phenolic acids. This study provides new insights into hormesis management for moringa plants and the influence of plant growth regulators on flavonoids and phenolic acid levels in moringa leaves under saline conditions.

An immobilized metal affinity (IMAC) adsorbent was prepared for selective enrichment of adenine type CKs, via grafting polymer chain pendant with iminodiacetic acid (IDA) from polydopamine (PDA)/polyethyleneimine (PEI)-coated magnetic graphene oxide (magGO) via surface-initiated-atom transfer radical polymerization (SI-ATRP). The prepared IMAC sorbent exhibited remarkable adsorption performances and good selectivity for adenine-type CKs and was utilized as a sorbent of magnetic solid-phase extraction (MSPE) for effective enrichment of four adenine-type CKs in bean sprouts. Under the optimized extraction conditions, an analytical method for four adenine type CKs in bean sprouts was established by combining the MSPE combined with ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The recoveries of the analytes were between 80.4 ± 1.9% and 114.6 ± 1.5% (n = 3). The limits of detection (LODs) range from 0.63 to 2.30 pg⋅mL -1 . The relative standard deviations of intra-day and inter-day were less than 12.6%. The established method was successfully applied to the selective extraction and sensitive detection of trace adenine-type CKs in plant samples., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Background: Symbiotic Methylobacterium strains comprise a significant part of plant microbiomes. Their presence enhances plant productivity and stress resistance, prompting classification of these strains as plant growth-promoting bacteria (PGPB). Methylobacteria can synthesize unusually high levels of plant hormones, called cytokinins (CKs), including the most active form, trans-Zeatin (tZ)., Results: This study provides a comprehensive inventory of 46 representatives of Methylobacterium genus with respect to phytohormone production in vitro, including 16 CK forms, abscisic acid (ABA) and indole-3-acetic acid (IAA). High performance-liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analyses revealed varying abilities of Methylobacterium strains to secrete phytohormones that ranged from 5.09 to 191.47 pmol mL -1 for total CKs, and 0.46 to 82.16 pmol mL -1 for tZ. Results indicate that reduced methanol availability, the sole carbon source for bacteria in the medium, stimulates CK secretion by Methylobacterium. Additionally, select strains were able to transform L-tryptophan into IAA while no ABA production was detected., Conclusions: To better understand features of CKs in plants, this study uncovers CK profiles of Methylobacterium that are instrumental in microbe selection for effective biofertilizer formulations., (© 2022. The Author(s).)

Plant hormones regulate numerous developmental and physiological processes. Abiotic stresses considerably affect production and distribution of phytohormones as the stress signal triggers. The homeostasis of plant hormones is controlled by their de novo synthesis and catabolism. The aim of this work was to analyse the contents of total and individual groups of endogenous cytokinins (CKs) as well as indole-3-acetic acid (IAA) in AtCKX overexpressing centaury plants grown in vitro on graded NaCl concentrations (0, 50, 100, 150, 200 mM). The levels of endogenous stress hormones including abscisic acid (ABA), salicylic acid (SA) and jasmonic acid (JA) were also detected. The elevated contents of total CKs were found in all analysed centaury shoots. Furthermore, increased amounts of all five CK groups, as well as enhanced total CKs were revealed on graded NaCl concentrations in non-transformed and AtCKX roots. All analysed AtCKX centaury lines exhibited decreased amounts of endogenous IAA in shoots and roots. Consequently, the IAA/bioactive CK forms ratios showed a significant variation in the shoots and roots of all AtCKX lines. In shoots and roots of both non-transformed and AtCKX transgenic centaury plants, salinity was associated with an increase of ABA and JA and a decrease of SA content., (© 2021. The Author(s).)

Cytokinins (CKs) are one class of important phytohormones widely investigated in most aspects of plant life. Similar to other phytohormones, CKs and their glycoconjugates are hydrophilic. Their ionization efficiencies for mass spectrometry (MS) detection are rather poor, whereas their retention and separation on reverse phase liquid chromatography (RPLC) are often unsatisfying. Chemical isotope labelling LC-MS analysis methods have been developed for most other phytohormones, enhancing their LC separations and quantitative sensitivity. However, there are currently no reports for chemical-labelled CKs. Here, we report a new chemical isotope labelling LC-MS analytical method for one-pot derivatization of CK bases and their glycoconjugates, based on differential benzylation labelling of the adenine skeleton of CKs with benzyl bromide and its deuterium isotope-labelled reagent. Benzylation alters the hydrophilicity of CKs and their glycoconjugates, improving their retention and separation on RPLC. The developed method demonstrated enhanced sensitivity, as the CKs and their glycoconjugates could be analysed with LODs within the range of 0.62-25.9 pg/mL. The method also demonstrated good intra- and inter-day precisions with standard deviations in the range of 1.9%-13.0%, and acceptable accuracy with recoveries in the range of 84.0%-119.9%. The developed method was employed on the quantitation of CKs in the fresh roots of Astragalus membranaceus collected from both fertilized and unfertilized fields. The significant impact that fertilizers had on endogenous CKs metabolism was observed. As such, monitoring endogenous CKs and their metabolites might be promising to control fertilizer abuse., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Plant hormones play important roles in plant growth and development and physiology, and in acclimation to environmental changes. The hormone signaling networks are highly complex and interconnected. It is thus important to not only know where the hormones are produced, how they are transported and how and where they are perceived, but also to monitor their distribution quantitatively, ideally in a non-invasive manner. Here we summarize the diverse set of tools available for quantifying and visualizing hormone distribution and dynamics. We provide an overview over the tools that are currently available, including transcriptional reporters, degradation sensors, and luciferase and fluorescent sensors, and compare the tools and their suitability for different purposes., (© 2020 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Arbuscular mycorrhizal symbiosis is a mutualistic interaction between most land plants and fungi of the glomeromycotina subphylum. The initiation, development and regulation of this symbiosis involve numerous signalling events between and within the symbiotic partners. Among other signals, phytohormones are known to play important roles at various stages of the interaction. During presymbiotic steps, plant roots exude strigolactones which stimulate fungal spore germination and hyphal branching, and promote the initiation of symbiosis. At later stages, different plant hormone classes can act as positive or negative regulators of the interaction. Although the fungus is known to reciprocally emit regulatory signals, its potential contribution to the phytohormonal pool has received little attention, and has so far only been addressed by indirect assays. In this study, using mass spectrometry, we analyzed phytohormones released into the medium by germinated spores of the arbuscular mycorrhizal fungus Rhizophagus irregularis. We detected the presence of a cytokinin (isopentenyl adenosine) and an auxin (indole-acetic acid). In addition, we identified a gibberellin (gibberellin A4) in spore extracts. We also used gas chromatography to show that R. irregularis produces ethylene from methionine and the α-keto γ-methylthio butyric acid pathway. These results highlight the possibility for AM fungi to use phytohormones to interact with their host plants, or to regulate their own development., Competing Interests: The authors have declared that no competing interests exist.

Thermally-processed coconut water often develop a commercially-undesirable pink color, thus, NMR, UPLC-HRMS, GC-MS analyses combined with chemometrics approach were applied to evaluate chemical variations in comparison to tender water (control) that could explain such color change. Chemometrics on negative ionization mode dataset showed trimeric and A-type dimeric procyanidins, and caffeoylshikimic acid as main identified secondary metabolites induced by processing, while, control water presented mainly cytokinin trans-zeatin riboside, procyanidin dimer, caffeoylshikimic acid and trihydroxy-octadecenoic acid. Processing increased long-chain saturated palmitic and stearic fatty acids contents, meanwhile NMR analysis showed a decline in primary metabolites content as sugars fructose and glucose, and short-chain organic acids. Among the results observed for thermally processed coconut water, the increase in oligomeric procyanidins as A-type dimer and trimer may be associated with pink color development as these are precursors of anthocyanin pigment and/or by enhancing color stability of anthocyanin solutions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Agronomically important traits often develop during the later stages of crop growth as consequences of various plant-environment interactions. Therefore, the temporal physiological states that change and accumulate during the crop's life course can significantly affect the eventual phenotypic differences in agronomic traits among crop varieties. Thus, to improve productivity, it is important to elucidate the associations between temporal physiological responses during the growth of different crop varieties and their agronomic traits. However, data representing the dynamics and diversity of physiological states in plants grown under field conditions are sparse. In this study, we quantified the endogenous levels of five phytohormones - auxin, cytokinins (CKs), ABA, jasmonate and salicylic acid - in the leaves of eight diverse barley (Hordeum vulgare) accessions grown under field conditions sampled weekly over their life course to assess the ongoing fluctuations in hormone levels in the different accessions under field growth conditions. Notably, we observed enormous changes over time in the development-related plant hormones, such as auxin and CKs. Using 3' RNA-seq-based transcriptome data from the same samples, we investigated the expression of barley genes orthologous to known hormone-related genes of Arabidopsis throughout the life course. These data illustrated the dynamics and diversity of the physiological states of these field-grown barley accessions. Together, our findings provide new insights into plant-environment interactions, highlighting that there is cultivar diversity in physiological responses during growth under field conditions., (© The Author(s) 2020. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Apple trees grafted on different rootstock types, including vigorous rootstock (VR), dwarfing interstock (DIR), and dwarfing self-rootstock (DSR), are widely planted in production, but the molecular determinants of tree branch architecture growth regulation induced by rootstocks are still not well known. In this study, the branch growth phenotypes of three combinations of 'Fuji' apple trees grafted on different rootstocks (VR: Malus baccata; DIR: Malus baccata/T337; DSR: T337) were investigated. The VR trees presented the biggest branch architecture. The results showed that the sugar content, sugar metabolism-related enzyme activities, and hormone content all presented obvious differences in the tender leaves and buds of apple trees grafted on these rootstocks. Transcriptomic profiles of the tender leaves adjacent to the top buds allowed us to identify genes that were potentially involved in signaling pathways that mediate the regulatory mechanisms underlying growth differences. In total, 3610 differentially expressed genes (DEGs) were identified through pairwise comparisons. The screened data suggested that sugar metabolism-related genes and complex hormone regulatory networks involved the auxin (IAA), cytokinin (CK), abscisic acid (ABA) and gibberellic acid (GA) pathways, as well as several transcription factors, participated in the complicated growth induction process. Overall, this study provides a framework for analysis of the molecular mechanisms underlying differential tree branch growth of apple trees grafted on different rootstocks., Competing Interests: The authors have declared that no competing interests exist.

In the context of future climate change new habitats will be threatened and unique species will be forced to develop different strategies to survive. Saxifraga longifolia Lapeyr. is an endemic species from the Pyrenees with a very particular habitat. We explored the capacity and strategies of S. longifolia plants to face different severities of drought stress under both natural conditions and controlled water stress followed by a re-watering period of 20 days. Our results showed a role for abscisic acid (ABA), salicylic acid (SA) and cytokinins (CKs) in plant survival from drought stress, and as the stress increased, ABA lost significance and SA appeared to be more associated with the response mechanisms. Moreover, photo-oxidative stress markers revealed that both xanthophyll cycles played a photoprotection role with a stronger participation of the lutein epoxide cycle as the stress was more intense. Severe drought decreased the maximum efficiency of photosystem II (F v /F m ) below 0.45, being this the limit to survive upon rewatering. Overall, our results proved different strategies of S. longifolia plants to cope with drought stress and suggested a F v /F m threshold to predict plant survival in high-mountain environments., (© 2020 Scandinavian Plant Physiology Society.)

Wheat awn plays a vital role in photosynthesis, grain production, and drought tolerance. However, the systematic identification or cloning of genes controlling wheat awn development is seldom reported. Here, we conducted a genome-wide association study (GWAS) with 364 wheat accessions and identified 26 loci involved in awn length development, including previously characterized B1, B2, Hd, and several rice homologs. The dominant awn suppressor B1 was fine mapped to a 125-kb physical interval, and a C 2 H 2 zinc finger protein Awn Length Inhibitor 1 (ALI-1) was confirmed to be the underlying gene of the B1 locus through the functional complimentary test with native awnless allele. ALI-1 expresses predominantly in the developing spike of awnless individuals, transcriptionally suppressing downstream genes. ALI-1 reduces cytokinin content and simultaneously restrains cytokinin signal transduction, leading to a stagnation of cell proliferation and reduction of cell numbers during awn development. Polymorphisms of four single nucleotide polymorphisms (SNPs) located in ALI-1 promoter region are diagnostic for the B1/b1 genotypes, and these SNPs are associated with awn length (AL), grain length (GL) and thousand-grain weight (TGW). More importantly, ali-1 was observed to increase grain length in wheat, which is a valuable attribute of awn on grain weight, aside from photosynthesis. Therefore, ALI-1 pleiotropically regulates awn and grain development, providing an alternative for grain yield improvement and addressing future climate changes., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

A number of scientific reports published to date contain data on endogenous levels of various phytohormones in potato (Solanum tuberosum L.) but a complete cytokinin profile of potato tissues, that would include data on all particular molecular forms of cytokinin, has still been missing. In this work, endogenous levels of all analytically detectable isoprenoid cytokinins, as well as the auxin indole-3-acetic acid (IAA), and abscisic acid (ABA) have been determined in shoots and roots of 30 day old in vitro grown potato (cv. Désirée). The results presented here are generally similar to other data reported for in vitro grown potato plants, whereas greenhouse-grown plants typically contain lower levels of ABA, possibly indicating that in vitro grown potato is exposed to chronic stress. Cytokinin N-glucosides, particularly N7-glucosides, are the dominant cytokinin forms in both shoots and roots of potato, whereas nucleobases, as the bioactive forms of cytokinins, comprise a low proportion of cytokinin levels in tissues of potato. Differences in phytohormone composition between shoots and roots of potato suggest specific patterns of transport and/or differences in tissue-specific metabolism of plant hormones. These results represent a contribution to understanding the hormonomics of potato, a crop species of extraordinary economic importance.

Cytokinins (CKs) are adenine derivatives that act as phytohormones. These signaling molecules control plant cell division and differentiation, organ growth, and senescence, and they orchestrate plant interactions with biotic and abiotic environments. While CKs are predominately recognized as plant-based substances, CKs have been found across different domains of life, including microorganisms, insects, mammals, and humans. In plants, CKs act at trace, often low femtomolar concentrations; therefore, sensitive and precise analytical techniques are required to accurately detect and quantify them from complex biological matrices. Here, we report the first comprehensive CK quantification method using a QExactive Orbitrap mass spectrometer in high-resolution with a parallel reaction monitoring (PRM)-based approach. The current method progresses upon multiple reaction monitoring (MRM) methods, previously used for CK profiling on triple quadrupole mass spectrometers. This method offers improved mass accuracy and the complete product ion mass spectra (MS/MS) for compound determination with increased specificity, and sensitivity comparable with triple quadrupole instruments. The presented PRM approach was successfully applied to quantify 32 CKs in several biological samples.

Cytokinin oxidase from Nipponbare (OsCKX4) was successfully displayed on the surface of E. coli cells by an ice nucleation protein from Pseudomonas borealis DL7 as an anchoring motif and a maltodextrin-binding protein(MBP) from E. coli as a solubility enhancer. The OsCKX4-displayed bacteria can be directly immobilized onto an electrode to selectively detect cytokinins, thus eliminating the need for enzyme extraction and purification. Direct electrochemistry of the cofactor FADH 2 in OsCKX4 has been achieved on an edge-plane pyrolytic graphite electrode (PGE) with a formal potential (E 0' ) of -0.45 V at pH 7.0 in phosphate buffer. With the addition of isopentenyladenine, the reduction peak current for FADH 2 decreased, and the oxidative peak current increased gradually. Therefore, a bacteria-OsCKX4-modified PGE has been developed for the detection of isopentenyladenine with a linear range of 1.0-11.0 μM and a lower limit of detection of 0.7 μM (S/N = 3). Slight interference was observed in the presence of other phytohormones, including brassinosteroid, abscisic acid, methylene jasminate and gibberellin. The proposed bacterial biosensor is stable, specific and simple and has great potential for applications that require the detection of cytokinins., (Copyright © 2019. Published by Elsevier B.V.)

A reliable, highly sensitive and highly selective method of high performance liquid chromatography associated with resonance Rayleigh scattering (HPLC-RRS) was developed to detect three cytokinins, namely, 6-benzylaminopurine (BA), kinetin (KT) and zeatin (ZT). In this work, Pd(ii) is added into the system to form ternary ion association complexes for the first time, which results in a lower limit of detection and extends the application of HPLC-RRS. The experimental conditions were optimized. In order to investigate the reaction mechanism, the ternary ion association complexes were characterized by ultraviolet-visible spectrophotometry, dynamic light scattering, scanning electron microscopy and density functional theory calculations. In a HAc-NaAc buffer solution (pH = 4.1), a ternary complex of cytokinin : Pd(ii) : EryB (1 : 1 : 2) was formed. The detection limits (S/N = 3) of BA, KT, and ZT were 0.9, 1.5 and 2.3 ng mL -1 , respectively. In addition, this method was applied for the simultaneous detection of cytokinins in real samples with satisfactory results.

Main Conclusion: The possible molecular mechanisms regulating strawberry fruit ripening were revealed by plant hormone quantification, exogenous hormone application, and RNA-sequencing. Fruit ripening involves a complex interplay among plant hormones. Strawberry is a model for studies on non-climacteric fruit ripening. However, the knowledge on how plant hormones are involved in strawberry ripening is still limited. To understand hormonal actions in the ripening process, we performed genome-wide transcriptome and hormonal analysis for the five major hormones (abscisic acid and catabolites, auxins, cytokinins, gibberellins, and ethylene) in achenes and receptacles (flesh) at different ripening stages of the woodland strawberry Fragaria vesca. Our results demonstrate that the pre-turning stage (a stage with white flesh and red achenes defined in this study) is the transition stage from immature to ripe fruits. The combinatorial analyses of hormone content, transcriptome data, and exogenous hormone treatment indicate that auxin is synthesized predominantly in achenes, while abscisic acid (ABA), bioactive free base cytokinins, gibberellins, and ethylene are mainly produced in receptacles. Furthermore, gibberellin may delay ripening, while ethylene and cytokinin are likely involved at later stages of the ripening process. Our results also provide additional evidence that ABA promotes ripening, while auxin delays it. Although our hormone analysis demonstrates that the total auxin in receptacles remains relatively low and unchanged during ripening, our experimental evidence further indicates that ABA likely enhances expression of the endoplasmic reticulum-localized auxin efflux carrier PIN-LIKES, which may subsequently reduce the auxin level in nucleus. This study provides a global picture for hormonal regulation of non-climacteric strawberry fruit ripening and also evidence for a possible mechanism of ABA and auxin interaction in the ripening process.

Hormonal dynamics after Plasmodiophora brassicae infection were compared in two Brassica napus cultivars-more resistant SY Alister and more sensitive Hornet, in order to elucidate responses associated with efficient defense. Both cultivars responded to infection by the early transient elevation of active cytokinins (predominantly cis -zeatin) and auxin indole-3-acetic acid (IAA) in leaves and roots, which was longer in Hornet. Moderate IAA levels in Hornet roots coincided with a high expression of biosynthetic gene nitrilase NIT1 (contrary to TAA1 , YUC8 , YUC9 ). Alister had a higher basal level of salicylic acid (SA), and it stimulated its production (via the expression of isochorismate synthase ( ICS1 )) in roots earlier than Hornet. Gall formation stimulated cytokinin, auxin, and SA levels-with a maximum 22 days after inoculation (dai). SA marker gene PR1 expression was the most profound at the time point where gall formation began, in leaves, roots, and especially in galls. Jasmonic acid (JA) was higher in Hornet than in Alister during the whole experiment. To investigate SA and JA function, SA was applied before infection, and twice (before infection and 15 dai), and JA at 15 dai. Double SA application diminished gall formation in Alister, and JA promoted gall formation in both cultivars. Activation of SA/JA pathways reflects the main differences in clubroot resistance.

Legume-Rhizobium symbiosis results in root nodules where rhizobia fix atmospheric nitrogen into plant usable forms in exchange for plant-derived carbohydrates. The development of these specialized root organs involves a set of carefully orchestrated plant hormone signalling. In particular, a spatio-temporal balance between auxin and cytokinin appears to be crucial for proper nodule development. We put together a construct that carried nuclear localized fluorescence sensors for auxin and cytokinin and used two photon induced fluorescence microscopy for concurrent quantitative 3-dimensional imaging to determine cellular level auxin and cytokinin outputs and ratios in root and nodule tissues of soybean. The use of nuclear localization signals on the markers and nuclei segmentation during image processing enabled accurate monitoring of outputs in 3D image volumes. The ratiometric method used here largely compensates for variations in individual outputs due to sample turbidity and scattering, an inherent issue when imaging thick root and nodule samples typical of many legumes. Overlays of determined auxin/cytokinin ratios on specific root zones and cell types accurately reflected those predicted based on previously reported outputs for each hormone individually. Importantly, distinct auxin/cytokinin ratios corresponded to distinct nodule cell types indicating a key role for these hormones in nodule cell type identity., (© 2018 The Authors Plant, Cell & Environment Published by John Wiley & Sons Ltd.)

Molecularly imprinted polymers prepared using traditional functional monomers and cross-linkers exhibit slow binding kinetics, low electrocatalytic activity and adsorption capacity. Herein, we report a new composite of ionic liquid-based graphene and molecularly imprinted polymer (IL-GR-MIP) with high electrocatalytic activity and adsorption capacity to construct an effective electrochemical sensor for 6-benzylaminopurine (6-BAP). Our objective was to enhance the efficiency of the sensor by incorporating more IL in the MIP framework. We synthesized IL-GR-MIP using ionic liquid 1-vinyl-3-butylimidazolium tetrafluoroborate (IL 1 ) as functional monomer, ionic liquid 1,4-butanediyl-3,3'-bis-l-vinylimidazolium dibromide (IL 2 ) as cross-linker, 6-BAP as template, and GR as supporter. IL-GR-MIP was characterized by Fourier transform infrared spectroscopy, thermal gravimetric analysis, Raman spectroscopy, X-ray photoelectron spectroscopy, and scanning electron microscope. Compared with GR-MIP composites based on methacrylic acid or IL 1 as functional monomer, N, N'-methylenebisacrylamide and ethylene glycol dimethacrylate as cross-linker, the IL-GR-MIP (prepared with ionic liquids as functional monomer and cross-linker) sensor exhibited highest peak current for 6-BAP. The results indicate the ability of IL 2 as cross-linker to enhance electrocatalytic activity and adsorption capacity for 6-BAP of IL-GR-MIP. Under the optimized conditions, the peak current of IL-GR-MIP sensor was linear to 6-BAP concentration in the range of 0.5-50 μM with a detection limit of 0.2 μM (S/N = 3). The IL-GR-MIP sensor exhibited good selectivity with the anti-interference ability of 1000-fold ascorbic acid in 6-BAP determination. Furthermore, we demonstrated practical applicability of IL-GR-MIP sensor in detecting 6-BAP in real samples with satisfactory results., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Adventitious shoot formation is an important technique for the propagation of economically important crops and for the regeneration of transgenic plants. Phytohormone treatment is required for the induction of adventitious shoots in most species. Whether adventitious shoots can be induced is determined by the balance between auxin and cytokinin (CK) levels. Much effort goes into determining optimum concentrations and combinations of phytohormones in each tissue used as explants and in each plant species. In ipecac, however, adventitious shoots can be induced on internodal segments in culture medium without phytohormone treatment. This allows the inherent plasticity of ipecac for cell differentiation to be evaluated. To induce adventitious shoots in ipecac, we cultured internodal segments at 24 °C under 15 µmol m -2 s -1 of light in a 14-h light/10-h dark cycle on phytohormone-free B5 medium solidified with 0.2% gellan gum for 5 weeks. To investigate phytohormone dynamics during adventitious shoot formation, we measured endogenous indole-3-acetic acid and CKs in the segments by liquid chromatography-tandem mass spectrometry LC-MS/MS. This method allows analysis of endogenous indole-3-acetic acid and CKs levels in a simple manner. It can be applied to investigate the dynamics of endogenous auxin and CK during organogenesis in other plant species.

We report a new method for histochemical localization of cytokinins (CKs) in plant tissues based on bromophenol blue/silver nitrate staining. The method was validated by immunohistochemistry using anti-trans-zeatin riboside antibody. Indole-3-acetic acid (auxin, IAA) was localized by anti-IAA antibody in plant tissues as a proof for IAA histolocalization. We used root sections, because they are major sites of CKs synthesis, and insect galls of Piptadenia gonoacantha that accumulate IAA. Immunostaining confirmed the presence of zeatin and sites of accumulation of IAA indicated by histochemistry. The colors developed by histochemical reactions in free-hand sections of plant tissues were similar to those obtained by thin layer chromatography (TLC), which reinforced the reactive sites of zeatin. The histochemical method for detecting CKs is useful for galls and roots, whereas IAA detection is more efficient for gall tissues. Therefore, galls constitute a useful model for validating histochemical techniques due to their rapid cell cycles and relatively high accumulation of plant hormones.

Rationale: Using mass spectrometry, the analysis of known metabolite structures has become feasible in a systematic high-throughput fashion. Nevertheless, the identification of previously unknown structures remains challenging, partially because many unidentified variants originate from known molecules that underwent unexpected modifications. Here, we present a method for the discovery of unknown metabolite modifications and conjugate metabolite isoforms in a high-throughput fashion., Methods: The method is based on user-controlled in-source fragmentation which is used to induce loss of weakly bound modifications. This is followed by the comparison of product ions from in-source fragmentation and collision-induced dissociation (CID). Diagonal MS 2 -MS 3 matching allows the detection of unknown metabolite modifications, as well as substructure similarities. As the method relies heavily on the advantages of in-source fragmentation and its ability to 'magically' elucidate unknown modification, we have named it inSourcerer as a portmanteau of in-source and sorcerer., Results: The method was evaluated using a set of 15 different cytokinin standards. Product ions from in-source fragmentation and CID were compared. Hierarchical clustering revealed that good matches are due to the presence of common substructures. Plant leaf extract, spiked with a mix of all 15 standards, was used to demonstrate the method's ability to detect these standards in a complex mixture, as well as confidently identify compounds already present in the plant material., Conclusions: Here we present a method that incorporates a classic liquid chromatography/mass spectrometry (LC/MS) workflow with fragmentation models and computational algorithms. The assumptions upon which the concept of the method was built were shown to be valid and the method showed that in-source fragmentation can be used to pinpoint structural similarities and indicate the occurrence of a modification., (Copyright © 2017 John Wiley & Sons, Ltd.)

Parallel determination of auxin and cytokinin levels within plant organs and tissues represents an invaluable tool for studies of their physiological effects and mutual interactions. Thanks to their different chemical structures, auxins, cytokinins and their metabolites are often determined separately, using specialized procedures of sample purification, extraction, and quantification. However, recent progress in the sensitivity of analytical methods of liquid chromatography coupled to mass spectrometry (LC-MS) allows parallel analysis of multiple compounds. Here we describe a method that is based on single step purification protocol followed by LC-MS separation and detection for parallel analysis of auxins, cytokinins and their metabolites in various plant tissues and cell cultures.

Background and Aims: The metabolism of cytokinins (CKs) and auxins in vascular plants is relatively well understood, but data concerning their metabolic pathways in non-vascular plants are still rather rare. With the aim of filling this gap, 20 representatives of taxonomically major lineages of cyanobacteria and algae from Cyanophyceae, Xanthophyceae, Eustigmatophyceae, Porphyridiophyceae, Chlorophyceae, Ulvophyceae, Trebouxiophyceae, Zygnematophyceae and Klebsormidiophyceae were analysed for endogenous profiles of CKs and auxins and some of them were used for studies of the metabolic fate of exogenously applied radiolabelled CK, [ 3 H]trans-zeatin (transZ) and auxin ([ 3 H]indole-3-acetic acid (IAA)), and the dynamics of endogenous CK and auxin pools during algal growth and cell division., Methods: Quantification of phytohormone levels was performed by high-performance or ultrahigh-performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-MS/MS, UHPLC-MS/MS). The dynamics of exogenously applied [ 3 H]transZ and [ 3 H]IAA in cell cultures were monitored by HPLC with on-line radioactivity detection., Key Results: The comprehensive screen of selected cyanobacteria and algae for endogenous CKs revealed a predominance of bioactive and phosphate CK forms while O- and N-glucosides evidently did not contribute greatly to the total CK pool. The abundance of cis-zeatin-type CKs and occurrence of CK 2-methylthio derivatives pointed to the tRNA pathway as a substantial source of CKs. The importance of the tRNA biosynthetic pathway was proved by the detection of tRNA-bound CKs during the course of Scenedesmus obliquus growth. Among auxins, free IAA and its oxidation catabolite 2-oxindole-3-acetic acid represented the prevailing endogenous forms. After treatment with [ 3 H]IAA, IAA-aspartate and indole-3-acetyl-1-glucosyl ester were detected as major auxin metabolites. Moreover, different dynamics of endogenous CKs and auxin profiles during S. obliquus culture clearly demonstrated diverse roles of both phytohormones in algal growth and cell division., Conclusions: Our data suggest the existence and functioning of a complex network of metabolic pathways and activity control of CKs and auxins in cyanobacteria and algae that apparently differ from those in vascular plants., (© The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company.)

Phytohormones, structurally diverse compounds, are involved in multiple processes within plants, such as controlling plant growth and stress response. Zn is an essential micronutrient for plants and its deficiency causes large economic losses in crops. Therefore, the purpose of this study was to analyse the role of phytohormones in the Zn-deficiency response of two economically important species, i.e. Lactuca sativa and Brassica oleracea. For this, these two species were grown hydroponically with different Zn-application rates: 10 μM Zn as control and 0.1 μM Zn as deficiency treatment and phytohormone concentration was determined by U-HPLC-MS. Zn deficiency resulted in a substantial loss of biomass in L. sativa plants that was correlated with a decline in growth-promoting hormones such as indole-3-acetic acid (IAA), cytokinins (CKs), and gibberellins (GAs). However these hormones increased or stabilized their concentrations in B. oleracea and could help to maintain the biomass in this species. A lower concentration of stress-signaling hormones such as ethylene precursor aminocyclopropane-1-carboxylic acid (ACC), abscisic acid (ABA), salicylic acid (SA) and jasmonic acid (JA) and also CKs might be involved in Zn uptake in L. sativa while a rise in GA4, isopentenyl adenine (iP), and ACC and a fall in JA and SA might contribute to a better Zn-utilization efficiency (ZnUtE), as observed in B. oleracea plants., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A high-throughput method was developed using liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) for the profiling and quantification of 43 phytohormones and their major metabolites, including auxins, abscisic acid, jasmonic acid, salicylic acid, cytokinins and gibberellins in a single sample extract. Considerable matrix effects (MEs) were observed (with most ME values in the range of 29%-84%, but maximum MEs of more than 115%, even up to 206%, existed) in sample extracts for most of the compounds studied. The application of the proposed binary solid-phase extraction using polymer anion and polymer cation exchange resins, was performed to purify 25 acidic and 18 alkaline phytohormones and their major metabolites prior to the LC-MS/MS analysis, which markedly reduced the MEs to acceptable levels, with ME values in the range of ±15%. Moreover, all of the isomers of cytokinins and their metabolites were fully separated on a sub-2μm particle C18 reverse-phase column with the optimized mobile phase consisting of methanol and 5mM ammonium formate. The method showed good linearity for all 43 analytes with regression coefficients (R(2))>0.991. Limits of detection ranged from 0.19 to 7.57 fmol for auxin, gibberellins, abscisic acid and their metabolites, 29.7 fmol for jasmonic acid, 18.1 fmol for salicylic acid, and from 0.03 to 0.31 fmol for cytokinins and their metabolites. The mean recoveries for all of the analytes were from 70.7 to 118.5%, and the inter-day precisions (n=6) were less than 18.7%, with intra-day precisions (n=6) within 25.4%. Finally, 20 compounds were successfully quantified in rice sample profiles using the proposed method, which will greatly facilitate the understanding of hormone-related regulatory networks that influence rice growth and development. To our knowledge, there are limited reports that measure this level of phytohormone species in rice samples using a single analysis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Cytokinins (CKs) are an important group of phytohormones. Their tightly regulated and balanced levels are essential for proper cell division and plant organ development. Here we report precise quantification of CK metabolites and other phytohormones in maize reproductive organs in the course of pollination and kernel maturation. A novel enzymatic activity dependent on NADP(+) converting trans-zeatin (tZ) to 6-(3-methylpyrrol-1-yl)purine (MPP) was detected. MPP shows weak anticytokinin properties and inhibition of CK dehydrogenases due to their ability to bind to an active site in the opposite orientation than substrates. Although the physiological significance of tZ side-chain cyclization is not anticipated as the MPP occurrence in maize tissue is very low, properties of the novel CK metabolite indicate its potential for utilization in plant in vitro tissue culture. Furthermore, feeding experiments with different isoprenoid CKs revealed distinct preferences in glycosylation of tZ and cis-zeatin (cZ). While tZ is preferentially glucosylated at the N9 position, cZ forms mainly O-glucosides. Since O-glucosides, in contrast to N9-glucosides, are resistant to irreversible cleavage catalyzed by CK dehydrogenases, the observed preference of maize CK glycosyltransferases to O-glycosylate zeatin in the cis-position might be a reason why cZ derivatives are over-accumulated in different maize tissues and organs., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Cytokinins play a critical role in controlling plant growth and development, but it is difficult to be determined in plant samples due to the extremely low concentration level of picomole/gram. So it is important for efficient sample preparation with selective enrichment and rapid separation for accurate analysis of cytokinins. Herein, a supramolecular perhydroxy-cucurbit[8]uril (PCB[8]) was fabricated into the Fe3O4 magnetic particles via chemical bonding assembly and magnetic perhydroxy-cucurbit[8]uril (MPC) materials were obtained. The MPC had good enrichment capability to cytokinins and the enrichment factors were more than 208. The interaction of MPC and cytokinins was investigated by adsorption test and density functional theory (DFT) calculation, the results showed that the main drive forces were the host-guest interaction and hydrogen-bonding interaction between the perhydroxy-cucurbit[8]uril with analytes. Combined with ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), the MPC was used as a sorbent of magnetic solid-phase extraction for the analysis of cytokinins in plant samples. A sensitive and selective UPLC-MS/MS method was developed with low detection limits of 0.14-0.32 ng/L for cytokinins analysis. Five cytokinins including zeatin riboside, meta-topolin, kinetin, kinetin riboside, and zip with 6.12-87.3 ng/kg were determined in the soybean sprout and Arabidopsis thaliana. The recoveries were in the range of 76.2-110% with relative standard deviations (n = 5) of 2.3-9.7%. On the basis of these results, magnetic perhydroxy-cucurbit[8]uril materials with selective enrichment capability have good potential on the analysis of ultratrace targets from complicated sample matrixes.

Plant beneficial microbes mediate biocontrol of diseases by interfering with pathogens or via strengthening the host. Although phytohormones, including cytokinins, are known to regulate plant development and physiology as well as plant immunity, their production by microorganisms has not been considered as a biocontrol mechanism. Here we identify the ability of Pseudomonas fluorescens G20-18 to efficiently control P. syringae infection in Arabidopsis, allowing maintenance of tissue integrity and ultimately biomass yield. Microbial cytokinin production was identified as a key determinant for this biocontrol effect on the hemibiotrophic bacterial pathogen. While cytokinin-deficient loss-of-function mutants of G20-18 exhibit impaired biocontrol, functional complementation with cytokinin biosynthetic genes restores cytokinin-mediated biocontrol, which is correlated with differential cytokinin levels in planta. Arabidopsis mutant analyses revealed the necessity of functional plant cytokinin perception and salicylic acid-dependent defence signalling for this biocontrol mechanism. These results demonstrate microbial cytokinin production as a novel microbe-based, hormone-mediated concept of biocontrol. This mechanism provides a basis to potentially develop novel, integrated plant protection strategies combining promotion of growth, a favourable physiological status and activation of fine-tuned direct defence and abiotic stress resilience.

Key Message: The current evidence of regulatory effect of smoke-water (SW) and karrikinolide (KAR(1)) on the concentrations of endogenous cytokinins in plants partly explain the basis for their growth stimulatory activity. Karrikinolide (KAR1) which is derived from smoke-water (SW) is involved in some physiological aspects in the life-cycle of plants. This suggests a potential influence on the endogenous pool (quantity and quality) of phytohormones such as cytokinins (CKs). In the current study, the effect of SW (1:500; 1:1000; 1:1500 v/v dilutions) and KAR1 (10(-7); 10(-8); 10(-9) M) applied during micropropagation of Eucomis autumnalis subspecies autumnalis on the ex vitro growth and CKs after 4 months post-flask duration was evaluated. The interactions of SW and KAR(1) with benzyladenine (BA), α-naphthaleneacetic acid (NAA) or BA+NAA were also assessed. Plants treated with SW (1:500) and KAR1 (10(-8) M) demonstrated superior growth in terms of the rooting, leaf and bulb sizes and fresh biomass than the control and plants treated with BA and BA+NAA. However, plant growth was generally inhibited with either SW (1:500) or KAR1 (10(-8) M) and BA when compared to BA (alone) treatment. Relative to NAA treatment, the presence of KAR(1) (10(-7) M) with NAA significantly increased the leaf area and fresh biomass. Both SW and KAR1-treated plants accumulated more total CKs, mainly isoprenoid-type than the control and NAA-treated plants. The highest CK content was also accumulated in SW (1:500) with BA+NAA treatments. Similar stimulatory effects were observed with increasing concentrations of KAR(1) and BA. The current findings establish that SW and KAR1 exert significant influence on the endogenous CK pools. However, the better growth of plants treated with SW and KAR1 treatments was not exclusively related to the endogenous CKs.

High-internal-phase-emulsion polymers (polyHIPEs) show great promise as solid-phase-extraction (SPE) materials because of the tremendous porosity and highly interconnected framework afforded by the high-internal-phase-emulsion (HIPE) technique. In this work, polyHIPE monolithic columns as novel SPE materials were prepared and applied to trace enrichment of cytokinins (CKs) from complex plant samples. The polyHIPE monoliths were synthesized via the in-situ polymerization of the continuous phase of a HIPE containing styrene (STY) and divinylbenzene (DVB) in a stainless column, and revealed highly efficient and selective enrichment ability for aromatic compounds. Under the optimized experimental conditions, a method using a monolithic polyHIPE column combined with liquid chromatography-electrospray tandem mass spectrometry (LC-MS-MS) was developed for the simultaneous extraction and sensitive determination of trans-zeatin (tZ), meta-topolin (mT), kinetin (K), and kinetin riboside (KR). The proposed method had good linearity, with correlation coefficients (R (2)) from 0.9957 to 0.9984, and low detection limits (LODs, S/N = 3) in the range 2.4-47 pg mL(-1) for the four CKs. The method was successfully applied to the determination of CKs in real plant samples, and obtained good recoveries ranging from 68.8 % to 103.0 % and relative standard deviations (RSDs) lower than 16 %.

Background: Bryophytes represent a very diverse group of non-vascular plants such as mosses, liverworts and hornworts and the oldest extant lineage of land plants. Determination of endogenous phytohormone profiles in bryophytes can provide substantial information about early land plant evolution. In this study, we screened thirty bryophyte species including six liverworts and twenty-four mosses for their phytohormone profiles in order to relate the hormonome with phylogeny in the plant kingdom., Methodology: Samples belonging to nine orders (Pelliales, Jungermanniales, Porellales, Sphagnales, Tetraphidales, Polytrichales, Dicranales, Bryales, Hypnales) were collected in Central and Northern Bohemia. The phytohormone content was analysed with a high performance liquid chromatography electrospray tandem-mass spectrometry (HPLC-ESI-MS/MS)., Principal Findings: As revealed for growth hormones, some common traits such as weak conjugation of both cytokinins and auxins, intensive production of cisZ-type cytokinins and strong oxidative degradation of auxins with abundance of a major primary catabolite 2-oxindole-3-acetic acid were pronounced in all bryophytes. Whereas apparent dissimilarities in growth hormones profiles between liverworts and mosses were evident, no obvious trends in stress hormone levels (abscisic acid, jasmonic acid, salicylic acid) were found with respect to the phylogeny., Conclusion: The apparent differences in conjugation and/or degradation strategies of growth hormones between liverworts and mosses might potentially show a hidden link between vascular plants and liverworts. On the other hand, the complement of stress hormones in bryophytes probably correlate rather with prevailing environmental conditions and plant survival strategy than with plant evolution.

The phytohormones, abscisic acid and cytokinin, once were thought to be present uniquely in plants, but increasing evidence suggests that these hormones are present in a wide variety of organisms. Few studies have examined fungi for the presence of these "plant" hormones or addressed whether their levels differ based on the nutrition mode of the fungus. This study examined 20 temperate forest fungi of differing nutritional modes (ectomycorrhizal, wood-rotting, saprotrophic). Abscisic acid and cytokinin were present in all fungi sampled; this indicated that the sampled fungi have the capacity to synthesize these two classes of phytohormones. Of the 27 cytokinins analyzed by HPLC-ESI MS/MS, seven were present in all fungi sampled. This suggested the existence of a common cytokinin metabolic pathway in fungi that does not vary among different nutritional modes. Predictions regarding the source of isopentenyl, cis-zeatin and methylthiol CK production stemming from the tRNA degradation pathway among fungi are discussed., (© 2015 by The Mycological Society of America.)

A rapid method was developed for determination of endogenous cytokinins (CKs) based on magnetic solid-phase extraction (MSPE) followed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). We illustrated the hydrophilic character of bare Fe3O4 nanoparticles that were directly used as a MSPE sorbent for rapid enrichment of endogenous CKs from complex plant extract. To the best of our knowledge, this is the first report of bare Fe3O4 directly used as efficient extraction sorbent to enrich target CKs based on hydrophilic interaction. Under the optimized conditions, a rapid, sensitive and high-throughput method for the determination of 16 CKs was established by combination of MSPE with UPLC-MS/MS. Good linearity was obtained with correlation coefficients (r) from 0.9902 to 0.9998. The limits of detection (LODs) and quantification (LOQs) ranged from 1.2 pg mL(-1) to 391.3 pg mL(-1) and 4.1 pg mL(-1) to 1304.3 pg mL(-1), respectively. 16 CKs could be successfully determined in spiked sample with 80.6-117.3% recoveries and the relative standard deviations (RSDs) were less than 16.6%. Finally, 10 endogenous CKs were successfully quantified in 50mg Oryza sativa sample using the developed MSPE-UPLC-MS/MS method., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We have developed a N6-dimethylallyladenine (cytokinin) dehydrogenase-based microbiosensor for real-time determination of the family of hormones known as cytokinins. Cytokinin dehydrogenase from Zea mays (ZmCKX1) was immobilised concurrently with electrodeposition of a silica gel film on the surface of a Pt microelectrode, which was further functionalized by free electron mediator 2,6-dichlorophenolindophenol (DCPIP) in supporting electrolyte to give a bioactive film capable of selective oxidative cleavage of the N6- side chain of cytokinins. The rapid electron shuffling between freely diffusible DCPIP and the FAD redox group in ZmCKX1 endowed the microbiosensor with a fast response time of less than 10 s. The immobilised ZmCKX1 retained a high affinity for its preferred substrate N6-(Δ2-isopentenyl) adenine (iP), and gave the miniaturized biosensor a large linear dynamic range from 10 nM to 10 µM, a detection limit of 3.9 nM and a high sensitivity to iP of 603.3 µAmM-1cm-2 (n = 4, R2 = 0.9999). Excellent selectivity was displayed for several other aliphatic cytokinins and their ribosides, including N6-(Δ2-isopentenyl) adenine, N6-(Δ2-isopentenyl) adenosine, cis-zeatin, trans-zeatin and trans-zeatin riboside. Aromatic cytokinins and metabolites such as cytokinin glucosides were generally poor substrates. The microbiosensors exhibited excellent stability in terms of pH and long-term storage and have been used successfully to determine low nanomolar cytokinin concentrations in tomato xylem sap exudates.

Biomass allocation between shoots and roots is an important strategy used by plants to optimize growth in various environments. Root to shoot mass ratios typically increase in response to high CO2, a trend particularly evident under abiotic stress. We investigated this preferential root growth (PRG) in Arabidopsis thaliana plants cultivated under low pH/high CO2 or low nitrogen (N)/high CO2 conditions. Previous studies have suggested that changes in plant hormone, carbon (C) and N status may be related to PRG. We therefore examined the mechanisms underlying PRG by genetically modifying cytokinin (CK) levels, C and N status, and sugar signaling, performing sugar application experiments and determining primary metabolites, plant hormones and expression of related genes. Both low pH/high CO2 and low N/high CO2 stresses induced increases in lateral root (LR) number and led to high C/N ratios; however, under low pH/high CO2 conditions, large quantities of C were accumulated, whereas under low N/high CO2 conditions, N was severely depleted. Analyses of a CK-deficient mutant and a starchless mutant, in conjunction with sugar application experiments, revealed that these stresses induce PRG via different mechanisms. Metabolite and hormone profile analysis indicated that under low pH/high CO2 conditions, excess C accumulation may enhance LR number through the dual actions of increased auxin and decreased CKs.

Identification of cytokinins in differentiated leaf cells has received little attention. We have carried out immunohistochemical localization of cytokinins in leaves of transgenic tobacco plants in which the level of increased due to induced in their roots the expression of ipt-gene controlling cytokinin synthesis. Immuno-labeling of cytokinins with the help of antibodies raised against zeatin riboside was characteristic of mesophyll cells. The label was localized in cytoplasm adjacent to cell walls and was absent in vacuoles. Immunohistochemical staining also revealed the presence of cytokinins in guard cells. Induction of cytokinin synthesis enhanced the immunohistochemical staining of both mesophyll cells and guard cells, which was accompanied by elevated stomatal conductance. The possibility of a direct effect of cytokinins on stomatal conductance and their indirect influence through photosynthesis in the mesophyll cells is discussed.

Cytokinins (CKs), a vital family of phytohormones, play important roles in the regulation of shoot and root development. However, the quantification of CKs in plant samples is frequently affected by the complex plant matrix. In the current study, we developed a simple, rapid and efficient hydrophilic interaction chromatography-solid phase extraction (HILIC-SPE) method for CKs purification. CKs were extracted by acetonitrile (ACN) followed by HILIC-SPE (silica as sorbents) purification. The extraction solution of plant samples could be directly applied to HILIC-SPE without solvent evaporation step, which simplified the analysis process. Moreover, with HILIC chromatographic retention mechanism, the hydrophobic co-extracted impurities were efficiently removed. Subsequently, CKs were separated by RPLC, orthogonal to the HILIC pretreatment process, and detected by tandem mass spectrometry. The method exhibits high specificity and recovery yield (>77.0%). Good linearities were obtained for all eight CKs ranging from 0.002 to 100ngmL(-1) with correlation coefficients (r) higher than 0.9927. The limits of detection (LODs, signal/noise=5) for the CKs were between 1.0 and 12.4pgmL(-1). Reproducibility of the method was evaluated by intra-day and inter-day measurements and the results showed that relative standard deviations (RSDs) were less than 10.5%. Employing this method, we successfully quantified six CKs in 20mg Oryza sativa leaves and the method was also successfully applied to Brassica napus (flower and leaves)., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Bacteria present in the rhizosphere of plants often synthesize phytohormones, and these signals can consequently affect root system development. In legumes, plants adapt to nitrogen starvation by forming lateral roots as well as a new organ, the root nodule, following a symbiotic interaction with bacteria collectively referred to as rhizobia. As cytokinin (CK) phytohormones were shown to be necessary and sufficient to induce root nodule organogenesis, the relevance of CK production by symbiotic rhizobia was questioned. In this study, we analyzed quantitatively, by liquid chromatography-tandem mass spectrometry, the production of 25 forms of CK in nine rhizobia strains belonging to four different species. All bacterial strains were able to synthesize a mix of CK, and bioactive forms of CK, such as iP, were notably found to be secreted in bacterial culture supernatants. Use of a mutant affected in extracellular polysaccharide (EPS) production revealed a negative correlation of EPS production with the ability to secrete CK. In addition, analysis of a nonnodulating Sinorhizobium meliloti strain revealed a similar pattern of CK production and secretion when compared with a related nodulating strain. This indicates that bacterially produced CK are not sufficient to induce symbiotic nodulation.

In Pinus radiata D. Don, the transition from the juvenile to the mature phase is characterized by a reduction in the tree's organogenic potential, which is usually reverted in breeding programs by reinvigoration procedures to enable vegetative propagation. In this work, we have determined the best culture conditions for in vitro reinvigoration of radiata pine buds, tested different cytokinin (CK) types [N⁶-benzyladenine (BA), meta-topolin (mT) and trans-zeatin] and concentrations (25 and 50 µM), and studied the effect of culture conditions on endogenous CK and indole-3-acetic acid (IAA) levels at different stages of the organogenic process. To this end, the levels of 43 CKs and IAA were determined in P. radiata buds before and during the reinvigoration process. When BA or mT was applied to the induction medium, we did not observe any significant increase or decrease in endogenous isoprenoid CK content. We also report for the first time the presence of O-glucosides in non-treated P. radiata explants from the field and remark the importance of O-glucosides as storage forms., (Copyright © Physiologia Plantarum 2012.)

The immunological adjuvant effect of silver nanoparticles (AgNPs) was investigated both in vitro and in vivo. The in vivo adjuvant effect of AgNPs was evaluated with model antigen ovalbumin (OVA) and bovine serum albumin (BSA) in mice by intraperitoneal and subcutaneous immunization. Serum antigen-specific IgG level significantly increased in AgNPs-treated mice comparing to the control group. AgNPs induced the increase of IgG1/IgG2a ratio and antigen-specific IgE, indicating that AgNPs elicited Th2-biased immune responses. By in vitro assay, the mechanism of adjuvant effect was explored. After 48h treatment with AgNPs, both the number of leukocytes and levels of cytokines TNF-α and IFN-γ in abdominal lavage fluid of mice increased. The expression of the major histocompatibility complex class II molecule on the surface of peritoneal macrophages significantly increased. AgNPs can be easily phagocytosed by peritoneal macrophages, while do not affect antigen uptake by the cell. We therefore conclude that AgNPs have significant adjuvant effect and the mechanism of this effect is mainly ascribed to the recruitment and activation of local leukocytes and especially macrophages. For the first time we found the remarkable adjuvant effect of AgNPs, and the result is beneficial for the future applications, especially in biomedicine., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

A method based on monolithic molecularly imprinted polymer enrichment combining with high performance liquid chromatography (mMIP-HPLC) detection was developed for the selective determination of trace cytokinins (CTKs) in plant samples. Monolithic molecularly imprinted polymer (mMIP) column was prepared in stainless steel tube by using kinetin as the template, methacrylic acid (MAA) as the functional monomer, ethylene glycol dimethacrylate (EGDMA) as the cross-linker, and toluene-dodecanol as the porogenic solvents. Compared with non-imprinted polymer (NIP) monolith, the prepared mMIP exhibited selective separation ability, good reproducibility and reusability, and high extraction efficiency in the separation and enrichment of the four CTKs. Under the optimized experimental conditions, mean recoveries were 91.9%, 80.0%, 87.5% and 50.2% for kinetin (K), kinetin glucoside (KR) , trans-zeatin (tZ) and meta-topolin (mT), respectively, with the corresponding RSDs less than 11.8%. The proposed mMIP-HPLC method was successfully applied in the separation and determination of the four cytokinins in different plant samples

Hormone crosstalk is pivotal in plant-pathogen interactions. Here, we report on the accumulation of cytokinins (CK) in rice seedlings after infection of blast fungus Magnaporthe oryzae and its potential significance in rice-M. oryzae interaction. Blast infection to rice seedlings increased levels of N(6)-(Δ(2)-isopentenyl) adenine (iP), iP riboside (iPR), and iPR 5'-phosphates (iPRP) in leaf blades. Consistent with this, CK signaling was activated around the infection sites, as shown by histochemical staining for β-glucuronidase activity driven by a CK-responsive OsRR6 promoter. Diverse CK species were also detected in the hyphae (mycelium), conidia, and culture filtrates of blast fungus, indicating that M. oryzae is capable of production as well as hyphal secretion of CK. Co-treatment of leaf blades with CK and salicylic acid (SA), but not with either one alone, markedly induced pathogenesis-related genes OsPR1b and probenazole-induced protein 1 (PBZ1). These effects were diminished by RNAi-knockdown of OsNPR1 or WRKY45, the key regulators of the SA signaling pathway in rice, indicating that the effects of CK depend on these two regulators. Taken together, our data imply a coevolutionary rice-M. oryzae interaction, wherein M. oryzae probably elevates rice CK levels for its own benefits such as nutrient translocation. Rice plants, on the other hand, sense it as an infection signal and activate defense reactions through the synergistic action with SA.

Caulogenesis in mature stone pine (Pinus pinea L.) cotyledons is promoted, to varying degrees depending on genotype, by exogenous application of the cytokinin (CK) benzyladenine (BA). In the present study, endogenous CK profiles of cotyledons from open-pollinated plants and two families of stone pine with widely differing organogenic capacities were monitored during caulogenesis and linked to previously characterized BA uptake and induction phases. Changes in levels of free bases, ribosides, ribotides and glucosides of both isoprenoid and aromatic CKs were followed. Before BA application, the pool of endogenous CKs in all sets of cotyledons was dominated by isoprenoid ribotides, but 1h after BA exposure, aromatic CKs (mainly active free bases and ribosides of topolins) accounted for more than 90% of the pool. BA N-glucosides were also observed, levels of which (and topolins) rose from 2d until the end of the (six-day) culture period. The CK profiles of the two selected pine families also differed, although the general trends were similar. During the first 6h, levels of BA and meta-topolin were highest in cotyledons from the family with the strongest caulogenic responses, while levels of ribotides and aromatic glucosides were highest in cotyledons from the other family., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

The effect of supplementing either meta-topolin (mT) or N(6)-benzyladenine (BA) requiring cultures with roscovitine (6-benzylamino-2-[1(R)-(hydroxymethyl)propyl]amino-9-isopropylpurine), a cyclin-dependent kinase (CDK) and N-glucosylation inhibitor, and INCYDE (2-chloro-6-(3-methoxyphenyl)aminopurine), an inhibitor of cytokinin (CK) degradation, on the endogenous CK profiles and physiology of banana in vitro was investigated. Growth parameters including multiplication rate and biomass were recorded after 42 days. Endogenous CK levels were quantified using UPLC-MS/MS while the photosynthetic pigment and phenolic contents were evaluated spectrophotometrically. The highest regeneration rate (93 %) was observed in BA + roscovitine while mT + INCYDE plantlets produced most shoots. Treatment with BA + roscovitine had the highest shoot length and biomass. Although not significant, there was a higher proanthocyanidin level in BA + roscovitine treatments compared to the control (BA). The levels of total phenolics and flavonoids were significantly higher in mT + roscovitine treatment than in the mT-treated regenerants. The presence of roscovitine and/or INCYDE had no significant effect on the photosynthetic pigments of the banana plantlets. Forty-seven aromatic and isoprenoid CKs categorized into nine CK-types were detected at varying concentrations. The presence of mT + roscovitine and/or INCYDE increased the levels of O-glucosides while 9-glucosides were higher in the presence of BA. Generally, the underground parts had higher CK levels than the aerial parts; however, the presence of INCYDE increased the level of CK quantified in the aerial parts. From a practical perspective, the use of roscovitine and INCYDE in micropropagation could be crucial in the alleviation of commonly observed in vitro-induced physiological abnormalities.

A capillary zone electrophoresis (CZE) method for separation of adenosine and N(6)-isopentenyladenosine (cytokinin) nucleotides was developed, optimized and validated. Aqueous solutions of several amino acids were evaluated as the background electrolyte constituents. Separation of six nucleotides in less than 20 min with high theoretical plate number (up to 400000 for isopentenyladenosine triphosphate) was achieved using a 100 mM sarcosine/ammonia buffer at pH 10.0. The detection limits of the CZE-UV method are in the low micromolar range (0.69-1.27 μmol L(-1)). Good repeatability of migration times (within 1.3%), peak areas (within 1.8%) and linearity (R(2)>0.999) was achieved over the concentration range 5-1000 μmol L(-1). The method was used to assay the activity of the recombinant Arabidopsis thaliana isopentenyltransferase 1 (AtIPT1). Baseline separation of isopentenylated nucleotides by CE-ESI-MS using a volatile buffer (30 mM ammonium formate; pH 10.0) was accomplished. The identities of the reaction products - isopentenyladenosine di- and triphosphate were confirmed by HPLC-QqTOF-MS. Dephosphorylation of ATP was observed as a parallel reaction., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Introduction: Cytokinins (CKs) are a group of plant hormones that play pivotal roles at low concentration in plant growth, development and regulatory pathways. In order to study the function, metabolism and signal transduction of CKs, high performance analytical techniques are required for determination of their endogenous levels., Objective: To develop a highly sensitive, selective and reliable method for identification and quantification of CKs by employing a tandem solid phase extraction (SPE)-online trapping-hydrophilic interaction chromatography (HILIC)-MS/MS method., Material and Methods: The extraction was performed firstly with tandem SPE containing a C(18) cartridge and a silica@C(8) /SO(3) H cartridge. After CKs were eluted from the silica@C(8)/SO(3) H cartridge, the desorption solvent was concentrated and redissolved in H(2)O and then injected into the online trapping-HILIC-MS/MS system with (Poly(MAA-co-EGDMA)) monolith as the trapping column. Subsequently, trapping, washing, desorption, separation and detection were accomplished automatically on the system., Results: Good linearities were obtained for eight cytokinins with correlation coefficients (R(2)) > 0.9964. The limits of detection (LOD; S:N = 3) for the targets ranged from 0.042 to 1.6 pg/mL. Reproducibility of the method was evaluated with intraday and interday relative standard deviations (RSDs) less than 13.4% and the recoveries ranged from 77.3% to 116.3%. The results showed that the LOD of the analytical method were at least one order of magnitude lower compared with other previously reported methods. Furthermore, only 20 mg of plant tissues were required for the quantitative analysis of the major CKs, which is, to the best of our knowledge, the smallest amount reported so far for the determination of endogenous CKs in plant tissues., Conclusion: The tandem SPE-online trapping-HILIC-MS/MS method developed in current study provides a powerful tool for the convenient and highly sensitive quantification of the major CKs in plant tissue., (Copyright © 2012 John Wiley & Sons, Ltd.)

Rationale: Mass spectrometry is effective for determination of cytokinins, which are bioactive compounds with an adenine-core structure. However, it is difficult to characterize any cytokinin compound without the relevant standard or known molecular structure information. With a limited number of standards, an in-source collision-induced dissociation (CID) method for characterization and classification of unknown cytokinins was described in this study., Methods: Cytokinins were separated by high-performance liquid chromatography and then analyzed by electrospray ionization ion trap mass spectrometry using an in-source CID technique and multiple reaction monitoring (MRM) mode., Results: Based on the properties of multi-stage fragmentation in mass spectrometry, naturally occurring cytokinins were classified into four types (zeatin, dihydrogen zeatin, isopentenyl adenine and benzyl adenine) by losing their conjugated sugar, sugar phosphate and other substituents in the source region. Following this technique, seven unknown cytokinins were characterized from roots of maize (Zea mays) without standards and one of them was finally confirmed to be cis-zeatin-riboside., Conclusions: An in-source CID technique combined with MRM mass spectrometry was developed to provide product ion information for identification of cytokinins and to afford guidance for the discovery of unknown cytokinins., (Copyright © 2012 John Wiley & Sons, Ltd.)