Analysis Of Cytokinins Research Articles

In vitro and ex vivo vegetative propagation and cytokinin profiles of Sceletium tortuosum (L.) N. E. Br.: a South African medicinal plant

Sceletium tortuosum is a South African protected species with tremendous value in traditional and modern medicine. The plants’ mesembrine-type alkaloids are potential therapeutics for a plethora of psychological, neurological and inflammatory disorders. In our in vitro and ex vivo studies, vegetative propagation and growth of this species were investigated. Cytokinin (CK) profiles were also explored. Shoot multiplication was induced on Murashige and Skoog (MS) medium supplemented with 2.5 µM indole-3-butyric acid (IBA). In vitro-generated shoots were inoculated on MS medium supplemented with 0, 2.5, 5.0 and 10.0 µM IBA or indole-3-acetic acid (IAA). Optimal rooting (55%), mean number of roots (3.80 ± 0.83) and new leaf pairs (4.65 ± 0.67) were achieved by 10.0 µM IBA. After greenhouse acclimatization, 45–90% of plantlets survived. All ex vivo shoot cuttings rooted well (90–100%). The highest mean number of roots (11.20 ± 1.37) and root length (57.18 ± 3.85 mm) were obtained by 5.0 µM IBA. Although spontaneous rooting was observed in both experiments, auxins enhanced multiple growth parameters. Cytokinin analyses of tissue-cultured (auxin-treated) and greenhouse (untreated) plants revealed higher cytokinin levels in vitro. These investigations provide rapid and efficient propagation techniques for Sceletium tortuosum which will be valuable to conservationists and pharmaceutical companies. Plant tissue culture and cuttings enabled rapid propagation of Sceletium tortuosum. Exogenous plant growth regulators were not essential for shoot multiplication, flowering and rooting. Auxins effectively improved growth parameters.

РІВЕНЬ ГЛЮКОЗИ ТА ГАММА-ГЛУТАМІЛТРАНСПЕПТИДАЗНА АКТИВНІСТЬ У КЛІТИНАХ ГЕПАТОЦИТАРНОГО ПОХОДЖЕННЯ ЗА ДІЇ ЕКСТРАКТІВ ТА ЦИТОКІНІНОВИХ ФРАКЦІЙ ЛІКАРСЬКИХ ГРИБІВ

Mushroom extracts show the multifunctional activity and have a wide range of applications for the treatment of various diseases, including cancer. However, the full composition of the compounds that produce macromycetes that exhibit antitumor properties has not yet been established. Impaired glucose metabolism and activation of gamma-glutamyltranspeptidase (GGT) in tumor cells may be a key marker of biochemical anaplasia in neoplasms. The aim of the study was to investigate the effect of crude extracts and cytokinin fractions isolated from the mycelial biomass of medicinal mushrooms on the biological properties of cells of hepatocyte-like cells of the HepG2 line (human hepatocellular carcinoma). The objects of the research were pure mushroom cultures of Hericium coralloides, Fomitopsis officinalis, Trametes (Coriolus) versicolor, Pleurotus ostreatus and Morchella esculenta. Cytokinin fractions from the extracts were isolated by centrifugation followed by fractionation and purification using ion exchange chromatography. Qualitative and quantitative analysis of cytokinins was performed by high-performance liquid chromatography. GGT activity was determined using the kit "Filisit" (Ukraine), glucose level – glucose oxidase method, with modifications for the cellular culture medium. The analysis of mycelial biomass of medicinal macromycetes revealed the presence of transzeatin, zeatin riboside, zeatin-O-glucoside and isopentenyladenine, that showed high activity in relation to cytokinin synthesis. Inhibition of glucose diffusion from the cultivation medium with the use of crude extracts and cytokinin fractions of medicinal mushroom and a decrease in GGT activity, more pronounced with the action of cytokinin fractions, compared with crude extracts, was noted. The difference between the effects of crude extracts and cytokinin fractions indicated the complex nature of the action of biologically active substances of medicinal mushroom. Presented results regarding the effects of crude extracts and cytokinin fractions of medicinal mushroom showed a normalizing effect on the main metabolic parameters which change in tumor cells, as a mechanism of biochemical anaplasia.

Improvement of Tillering and Grain Yield by Application of Cytokinin Derivatives in Wheat and Barley

Three cytokinin derivatives (CKd) designated as RR-G, RR-O, and RR-V applied by foliar spraying at tillering, and one compound previously described as a cytokinin antagonist (CKa) designated as RR-P applied as a seed coating were tested in winter wheat and spring barley in field trial experiments. The aim of the study was to examine the influence of the compounds that were tested on the number of productive tillers, grain yield, and endogenous CK content. With the exception of the compound RR-V, the measured parameters clearly showed the stimulatory effects of CKd on tillering and grain yield in spring barley and winter wheat. The RR-V showed a stimulatory effect on the number of productive tillers and yield in spring barley, but not in winter wheat. Although in winter wheat CKa stimulated both the number of productive tillers and the grain yield, there was an inhibitory effect in terms of the number of productive tillers observed in spring barley. The results of the endogenous cytokinin analysis suggested, among others, the importance of the role of isopentenyl-adenine types of cytokinins in the tillering of spring barley. In conclusion, the cytokinin derivative compounds with an agonistic or antagonistic role showed strong potential for application in the future development of plant growth regulators.

Cytokinin dehydrogenase: a genetic target for yield improvement in wheat.

SummaryThe plant hormone group, the cytokinins, is implicated in both qualitative and quantitative components of yield. Cytokinins have opposing actions in shoot and root growth—actions shown to involve cytokinin dehydrogenase (CKX), the enzyme that inactivates cytokinin. We revise and provide unambiguous names for the CKX gene family members in wheat, based on the most recently released wheat genome database, IWGSC RefSeq v1.0 & v2.0. We review expression data of CKX gene family members in wheat, revealing tissue‐specific gene family member expression as well as sub‐genome‐specific expression. Manipulation of CKX in cereals shows clear impacts on yield, root growth and orientation, and Zn nutrition, but this also emphasizes the necessity to unlink promotive effects on grain yield from negative effects of cytokinin on root growth and uptake of mineral nutrients, particularly Zn and Fe. Wheat is the most widely grown cereal crop globally, yet is under‐research compared with rice and maize. We highlight gaps in our knowledge of the involvement of CKX for wheat. We also highlight the necessity for accurate analysis of endogenous cytokinins, acknowledging why this is challenging, and provide examples where inadequate analyses of endogenous cytokinins have led to unjustified conclusions. We acknowledge that the allohexaploid nature of bread wheat poses challenges in terms of uncovering useful mutations. However, we predict TILLING followed by whole‐exome sequencing will uncover informative mutations and we indicate the potential for stacking mutations within the three genomes to modify yield components. We model a wheat ideotype based on CKX manipulation.

Green spathe of peace lily (Spathiphyllum wallisii): An assimilate source for developing fruit

Peace lily (Spathiphyllum wallisii Regel) is a herbaceous, commercially important ornamental plant characterized by greening of its originally white spathe during a post-anthesis period and fruit development. It is a useful model plant for gaining insight into source–sink relationship. To clarify what is the triggering signal for spathe greening and its potential physiological role, the greening process has been examined in fertilized vs unfertilized and decapitated (with spadix removed) plants. Results showed that spathe of fertilized plants is viable for a longer period of time and greening process is more intensive in comparison with decapitated or unfertilized plants. Greening process comprised chloroplast differentiation as well as chlorophyll accumulation, and resulted in the photosynthetically active organs. Photosynthesis performance index (PIABS) of spathe in fertilized flowers was significantly higher than that in unfertilized and decapitated plants, indicating its better overall photosynthetic performance. Spathe removal during fruit development revealed approximately 30% smaller fruits in comparison with intact ones, implicating that green spathe contribute significantly as assimilate source for developing fruits. Cytokinin analysis showed that t-Z-type cytokinins were the most abundant in the spadix (t-Z and t-ZR) and the greening spathe (t-ZR), while overall cytokinin pool was significantly lower in the spathe of the decapitated plants. Exogenous treatment of spathe in decapitated plants with t-Z enhanced the intensity of greening. We may conclude that spathe greening in peace lily is genetically programmed process that may be mediated by fructification and, in part, by cytokinins originated in the developing fruit which was confirmed herein for the first time.

EXPRESSION PROFILING OF SELECTED MICRO RNAs COUPLED WITH MOLECULAR AND BIOCHEMICAL ANALYSES OF ASIATICOSIDES IN CENTELLA ASIATICA (L.)URB IN VITRO

Objective: Centella asiatica (L.) Urb from Umbelliferae is a potential source of secondary metabolites having immense medicinal value. Asiaticoside is the major therapeutic compound. In the present study, Identification of a possible relationship between concentration/transcript level expression of asiaticoside and concentrations of growth hormones at different growth stages was observed. The current study includes molecular and biochemical evaluation of stress generated in C. asiatica at different time intervals in vitro.
 Methods: The enhancement in auxin, cytokinin and final asiaticoside content were determined using immunoassay kits for auxin, cytokinin and HPLC analysis respectively. Transcript level expression at different growth phases was carried out using real-time RT-PCR. For isolation of stress-related miRNAs, reverse transcription of total RNA using miScript II RT Kit PCR System was carried out as per instructions. The differential expression of five selected miRNAs was done by Real-Time RT-PCR. The analysis of stress in vitro was done by quantification of Hydrogen Peroxide (H2O2), total phenolics and total antioxidants by H2O2 assay kit, total antioxidant assay kit and Folin Ciocalteau reagent respectively. The final asiaticoside content was determined by HPLC.
 Results: Differential expression of key genes involved in asiaticoside pathway showed significantly higher transcript expression, which is in correlation with the final asiaticoside content. The enhanced expression of miRNAs and the analysis of H2O2, total antioxidant capacity and total phenolics are suggestive of generation of oxidative stress under controlled conditions.
 Conclusion: The present study shows a direct correlation between oxidative stress and transcript/phytochemical estimation of asiaticoside content under in vitro conditions.

Setting-up a fast and reliable cytokinin biosensor based on a plant histidine kinase receptor expressed in Saccharomyces cerevisiae

Cytokinins (CK) have been extensively studied for their roles in plant development. Recently, they also appeared to ensure crucial functions in the pathogenicity of some bacterial and fungal plant pathogens. Thus, identifying cytokinin-producing pathogens is a prerequisite to gain a better understanding of their role in pathogenicity. Taking advantage of the cytokinin perception properties of Malus domestica CHASE Histidine Kinase receptor 2 (MdCHK2), we thereby developed a selective and highly sensitive yeast biosensor for the application of cytokinin detection in bacterial samples. The biosensor is based on the mutated sln1Δ Saccharomyces cerevisiae strain expressing MdCHK2. The biosensor does not require any extraction or purification steps of biological samples, enabling cytokinin analysis directly from crude bacterial supernatants. For the first time, the production of cytokinin was shown in the well-known plant pathogenic bacteria Erwinia amylovora and was also revealed in human pathogens Staphylococcus aureus and Streptococcus agalactiae. Importantly, this biosensor was shown to be an efficient tool for unraveling certain steps in cytokinin biosynthesis by micro-organisms since this it was successfully used to unveil the role of ygdH22, a LOG-like gene, that is probably involved in cytokinin biosynthesis pathway in Escherichia coli. Overall, we demonstrated that our biosensor displays several advantages including time- and cost-effectiveness by allowing a rapid and specific detection of cytokinins in bacterial supernatants These results also support its scalability to high-throughput formats.

Hormonal interactions during early physiological partenocarpic fruitlet abscission in persimmon (Diospyros Kaki Thunb.) ‘Triumph’ and ‘Shinshu’ cultivars

Fruitlet abscission is typical to many fruit trees, including persimmon, (Diopyros kaki Thunb.), and often results in unstable fruit production and yield. We documented massive parthenocarpic fruitlet abscission in the persimmon cultivars 'Triumph' and 'Shinshu', which initiated soon after anthesis and peaked at 45 days post anthesis. In contrast, abscission of mature fruitlets 45–65 days after anthesis was minor. The early parthenocarpic fruitlet abscission in 'Shinshu' and 'Triumph' took place in the abscission zone located at the calyx-pedicel junction. Differentiation of the abscission layer occurred during flower development and preceded fruitlet abscission. Endogenous analysis of gibberellins, cytokinins and auxin levels in the abscission zone of 'Triumph' and 'Shinshu' during the developmental stages preceding abscission revealed a reduction in cytokinin endogenous levels in both cultivars. In addition, a decline in GA3 and IAA levels was found in 'Triumph' and 'Shinsu' respectively during flower development. Gibberellin, auxin and cytokinin application to inhibit early fruitlet abscission were analyzed and gibberellin treatment exhibited the highest and most consistent inhibition effect in 'Triumph' and 'Shinshu'. An increase in endogenous levels of gibberellins, cytokinins and auxin in the flower abscission zone was found following GA application in both cultivars. Our results suggest that gibberellin application inhibits early fruitlet abscission by elevating cytokinins, and auxin levels and that a reduction in gibberellins, cytokinins and auxin levels may induce abscission during parthenocarpic fruitlet development in 'Shinshu' and 'Triumph' cultivars.

English

The chemical composition of olive leaves and roots of Chemlali variety was evaluated regarding the contents of amino acids and endogenous phytohormones under foliar application of biofertilizers (F1: rich in nitrogen (N), phosphorus (P), potassium (K); F2: rich in calcium (Ca); F3: application of F1 and F2). The results of this study demonstrated significant effects of tested fertilizers on the concentrations of amino acids. Glutamic acid and asparagine had the highest concentrations in leaves and the lowest concentrations in roots under F1 and F3. Regarding the concentrations of endogenous phytohormones, high contents of indole-3-acetic acid and salicylic acid were found in leaves and roots under the application of F1 biofertilizer. Jasmonic acid (JA) was of higher concentration in root than leaves, while abscisic acid occurred in lower concentration. Moreover, the analysis of isoprenoid cytokinins by ultra-performance liquid-chromatography tandem mass spectrometry (UPLC-MS/MS) showed that these cytokinins were improved and became more abundant in roots under foliar treatments. Particularly, Zeatin-N7-glucoside and Dihydrozeatin-N9-Glucoside were enhanced under F1 and F3. The results of this study suggest that foliar application of biological fertilizers may improve physiological status of olive trees throughout the enhancement of some phytohormones and amino acids. Key words: Foliar fertilization, olive trees, amino acids, indole-3-acetic acid, isoprenoid cytokinins.

High-Resolution Cell-Type Specific Analysis of Cytokinins in Sorted Root Cell Populations of Arabidopsis thaliana.

We describe a method combining fluorescence-activated cell sorting (FACS) with one-step miniaturized isolation and accurate quantification of cytokinins (CKs) using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to measure these phytohormones in specific cell types of Arabidopsis thaliana roots. The methodology provides information of unprecedented resolution about spatial distributions of CKs, and thus should facilitate attempts to elucidate regulatory networks involved in root developmental processes.

High-throughput quantification of more than 100 primary- and secondary-metabolites, and phytohormones by a single solid-phase extraction based sample preparation with analysis by UHPLC-HESI-MS/MS.

BackgroundPlant metabolites are commonly functionally classified, as defense- or growth-related phytohormones, primary and specialized metabolites, and so forth. Analytical procedures for the quantifications of these metabolites are challenging because the metabolites can vary over several orders of magnitude in concentrations in the same tissues and have very different chemical characteristics. Plants clearly adjust their metabolism to respond to their prevailing circumstances in very sophisticated ways that blur the boundaries among these functional or chemically defined classifications. But if plant biologists want to better understand the processes that are important for a plant’s adaptation to its environment, procedures are needed that can provide simultaneous quantifications of the large range of metabolites that have the potential to play central roles in these adjustments in a cost and time effective way and with a low sample consumption.ResultsHere we present a method that combines well-established methods for the targeted analysis of phytohormones, including jasmonates, salicylic acid, abscisic acid, gibberellins, auxins and cytokinins, and extends it to the analysis of inducible and constitutive defense compounds, as well as the primary metabolites involved in the biosynthesis of specialized metabolites and responsible for nutritional quality (e.g., sugars and amino acids). The method is based on a single extraction of 10–100 mg of tissue and allows a broad quantitative screening of metabolites optimized by their chemical characteristics and concentrations, thereby providing a high throughput analysis unbiased by the putative functional attributes of the metabolites. The tissues of Nicotiana attenuata which accumulate high levels of nicotine and diterpene glycosides, provide a challenging matrix that thwarts quantitative analysis; the analysis of various tissues of this plant are used to illustrate the robustness of the procedure.ConclusionsThe method described has the potential to unravel various, until now overlooked interactions among different sectors of plant metabolism in a high throughput manner. Additionally, the method could be particularly beneficial as screening method in forward genetic approaches, as well as for the investigation of plants from natural populations that likely differ in metabolic traits.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-016-0130-x) contains supplementary material, which is available to authorized users.

Efficient and Selective Enrichment of Ultratrace Cytokinins in Plant Samples by Magnetic Perhydroxy-Cucurbit[8]uril Microspheres.

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.

Extra- and intracellular distribution of cytokinins in the leaves of monocots and dicots

The plant hormones cytokinins are a convenient target of genetic manipulations that bring benefits in biotechnological applications. The present work demonstrates the importance of the subcellular compartmentalization of cytokinins on the model dicot plant Arabidopsis thaliana and monocot crop Hordeum vulgare. The method of protoplast and vacuole isolation combined with precise cytokinin analysis and recovery assay of a vacuolar marker protein were used to quantify the contents of individual cytokinin forms in the leaf extracellular space, cell interior and vacuole. The data obtained for wild type plants and in each case a specific mutant line allow comparing the effect of genetic manipulations on the hormone distribution and homeostatic balance of cytokinins in the modified plants.

Cytokinin dynamics in differently senescing laminae of Phragmites australis plants grown in different habitats

Common reed is a cosmopolitan species, which occurs also in South Africa. At a bay of the Baltic Sea stands of tall, large-leafed reed (TR) and stands of short, small-leafed reed (SR) were found. While TR grew where deep soil with much humus and phosphate was available, SR was found growing on shallow soil, which was often of relatively high salinity. As changes in coloration and chlorophyll analyses showed, SR laminae senesced earlier than did TR laminae. The differences in growth and senescence were eliminated by transplanting TR and SR into garden earth. As literature data from other species show, soil components like phosphate and NaCl can influence levels of the antisenescence hormones cytokinins. Our aim was to find correlations between cytokinin levels and the vitality in reed laminae from natural sites. We used ELISAs for cytokinin analysis and completed the investigations by LC-MS determination of 17 cytokinins and cytokinin conjugates in laminae of HR and SR grown near Prague. From July to autumn the content of trans-zeatin, dihydrozeatin and their 9-ribosides decreased in third laminae under panicles at TR on an average to 1.3nmol trans-zeatin-9-riboside equivalents kg−1 fresh mass and at SR to 0.7 (8 sample pairs). In autumn SR laminae had also lower levels of N6-(Δ2-isopentenyl)adenine and its 9-riboside and probably also of trans-zeatin-O-glucoside and cis-zeatin-9-riboside-O-glucoside compared to TR laminae. The putatively inactive or weakly active cis-zeatin compound increased during the summer to very high quantities. After isolation of laminae, the content of cytokinins declined and cytokinin application delayed yellowing. Contrary to some other examples, our results are in concordance with the rule that endogenous cytokinins retard leaf senescence and help thus to explain the senescence differences in TR and SR.

Analysis of 17 cytokinins in rice by solid phase extraction purification and liquid chromatography-tandem mass spectrometry

A method was developed for the determination of 17 cytokinins in rice by solid phase extraction and liquid chromatography-tandem mass spectrometry. The rice samples were cryogenically ground under liquid nitrogen and extracted with methanol-water (80:20, v/v) at 4 °C for 16 h. The supernatant was purified on a column packed with polymer cation exchange resin (PCX). The samples were transported and eluted on an analytical column ZORBAX Extend-C18 (100 mm x 2.1 mm, 1.8 μm) by methanol and 5 mmol/L ammonium formate aqueous solution. All the analytes were detected in selected reaction monitoring (SRM) mode under positive electrospray ionization (ESI+) and quantified by external standard method. The separation conditions were optimized in order to achieve the sufficient separation for the several isomers of cytokinins, such as trans-zeatin-7-glucoside (tZ7G), trans-zeatin-9-glucoside (tZ9G), and trans-zeatin-O-glucoside (tZOG). Moreover, the extraction efficiency of different extraction solvents and clean-up effects of PCX cartridge for each analyte were further investigated. The results showed that the correlation coefficients were not less than 0.9984 in the linear range, and the limits of detection were ranged from 0.01 to 0.05 ng/g. The mean recoveries of the 17 cytokinins at three spiked levels of 0.2, 1 and 5 ng/g were from 60.2% to 125.4% with the relative standard deviations (RSDs) of 5.4%-29.7% (n = 6). Finally, five endogenous cytokinins were successfully quantified in real sample, and their contents were between 0.02 and 0.93 ng/g. It means that this method is reliable for quantitative analysis of cytokinins in rice.

Quantitative Analysis of Cytokinins in Plants by High Performance Liquid Chromatography: Electronspray Ionization Ion Trap Mass Spectrometry

The present paper introduces a highly sensitive and selective method for simultaneous quantification of 12 cytokinins (free form and their conjugates). The method includes a protocol of extraction with methanol/water/formic acid (15/4/1, v/v/v) to the micro-scale samples, pre-purification with solid phase extraction (SPE) cartridges of the extracts, separation with a high performance liquid chromatography (HPLC) and detection by an electrospray ionization ion trap mass spectrometry (ESI-Ion trap-MS) system in a consecutive ion monitoring (CRM) mode at the three stage fragmentation of mass spectrometry (MS(3) ). The lowest detection level of the cytokinins of the method reaches 0.1-2.0 pg with a very wide range of linear regression from 1-512 pg, at the coefficient factors of 0.98-0.99. The feasibility of this method has been proven in the application of the method to the analysis of the trace-amount contents of cytokinins in the micro-scale samples of various types of plant materials, such as aerial parts of rice and poplar leaves etc. 12 endogenous cytokinins had been identified and quantified in the plant tissues, with an acceptable relatively higher recovery rate from 40% to 70%.

Endogenous cytokinins in Cocos nucifera L. in vitro cultures obtained from plumular explants

Auxin induces in vitro somatic embryogenesis in coconut plumular explants through callus formation. Embryogenic calli and non-embryogenic calli can be formed from the initial calli. Analysis of endogenous cytokinins showed the occurrence of cytokinins with aromatic and aliphatic side chains. Fourteen aliphatic cytokinins and four aromatic cytokinins were analysed in the three types of calli and all the cytokinins were found in each type, although some in larger proportions than others. The most abundant cytokinins in each type of callus were isopentenyladenine-9-glucoside, zeatin-9-glucoside, zeatin riboside, isopentenyladenine riboside, dihydrozeatin and dihydrozeatin riboside in decreasing order. Total cytokinin content was compared between the three types of calli, and it was found to be lower in embryogenic calli compared to non-embryogenic calli or initial calli. The same pattern was observed for individual cytokinins. When explants were cultured in media containing exogenously added cytokinins, the formation of embryogenic calli in the explants was reduced. When 8-azaadenine (an anticytokinin) was added the formation of embryogenic calli and somatic embryos was increased. These results suggest that the difference in somatic embryo formation capacity observed between embryogenic calli and non-embryogenic calli is related to their endogenous cytokinin contents.

AtSOFL1 and AtSOFL2 Act Redundantly as Positive Modulators of the Endogenous Content of Specific Cytokinins in Arabidopsis

BackgroundAlthough cytokinins have been known for decades to play important roles in the regulation of plant growth and development, our knowledge of the regulatory mechanism of endogenous content of specific cytokinins remains limited.Methodology/Principal FindingsHere, we characterized two SOB five-like (SOFL) genes, AtSOFL1 and AtSOFL2, in Arabidopsis (Arabidopsis thaliana) and showed that they acted redundantly in regulating specific cytokinin levels. Analysis of the translational fusion AtSOFL1:AtSOFL1-GUS and AtSOFL2:AtSOFL2-GUS indicated that AtSOFL1 and AtSOFL2 exhibited similar expression patterns. Both proteins were predominantly expressed in the vascular tissues of developing leaves, flowers and siliques, but barely detectable in roots and stems. Overexpression of either AtSOFL1 or AtSOFL2 led to increased cytokinin content and obvious corresponding mutant phenotypes for both transgenic seedlings and adult plants. In addition, overexpression and site-directed mutagenesis experiments demonstrated that the SOFL domains are necessary for AtSOFL2's overexpression phenotypes. Silencing or disrupting either AtSOFL1 or AtSOFL2 caused no obvious developmental defects. Endogenous cytokinin analysis, however, revealed that compared to the wild type control, the SOFL1-RNAi62 sofl2-1 double mutant accumulated lower levels of trans-zeatin riboside monophosphate (tZRMP) and N6-(Δ2-isopentenyl)adenosine monophosphate (iPRMP), which are biosynthetic intermediates of bioactive cytokinins. The double mutant also displayed decreased response to exogenous cytokinin in both callus-formation and inhibition-of-hypocotyl-elongation assays.Conclusions/SignificanceTaken together, our data suggest that in plants AtSOFL1 and AtSOFL2 work redundantly as positive modulators in the fine-tuning of specific cytokinin levels as well as responsiveness.

Thermoperiod affects the diurnal cycle of nitrate reductase expression and activity in pineapple plants by modulating the endogenous levels of cytokinins

Nitrate reductase (NR, EC 1.6.6.1) activity in higher plants is regulated by a variety of environmental factors and oscillates with a characteristic diurnal rhythm. In this study, we have demonstrated that the diurnal cycle of NR expression and activity in pineapple (Ananas comosus, cv. Smooth Cayenne) can be strongly modified by changes in the day/night temperature regime. Plants grown under constant temperature (28 degrees C light/dark) showed a marked increase in the shoot NR activity (NRA) during the first half of the light period, whereas under thermoperiodic conditions (28 degrees C light/15 degrees C dark) significant elevations in the NRA were detected only in the root tissues at night. Under both conditions, increases in NR transcript levels occurred synchronically about 4 h prior to the corresponding elevation of the NRA. Diurnal analysis of endogenous cytokinins indicated that transitory increases in the levels of zeatin, zeatin riboside and isopentenyladenine riboside coincided with the accumulation of NR transcripts and preceded the rise of NRA in the shoot during the day and in the root at night, suggesting these hormones as mediators of the temperature-induced modifications of the NR cycle. Moreover, these cytokinins also induced NRA in pineapple when applied exogenously. Altogether, these results provide evidence that thermoperiodism can modify the diurnal cycle of NR expression and activity in pineapple both temporally and spatially, possibly by modulating the day/night changes in the cytokinin levels. A potential relationship between the day/night NR cycle and the photosynthetic pathway performed by the pineapple plants (C(3) or CAM) is also discussed.

Research Progress in Cytokinins Analysis

Abstract Quantitative trace analysis of cytokinins (CTKs) is of great importance for illustrating the signal transduction and regulation mechanism of CTKs in plants. In this article, analytical methods including immunoassay, gas chromatography, high performance liquid chromatography, capillary electrophoresis, and electroanalytical technique for CTKs detection are all reviewed. Related sample pretreatment methods are introduced. The future development trend in CTKs analysis is also discussed.