Changes in the distribution of endogenous hormones in Phyllostachys edulis 'Pachyloen' during bamboo shooting.

In this study, we investigated the changes in the distribution and regulation of endogenous hormones in Phyllostachys edulis 'Pachyloen' during bamboo shooting. Enzyme-linked immunosorbent assay was used to measure the mass fractions of indole-3-acetic acid (IAA), gibberellic acid (GA), zeatin riboside (ZR), and abscisic acid (ABA) in rhizomes, shoots, and maternal bamboo organs during shoot sprouting, shoot growth, and new-bamboo formation. Measurements were compared among bamboo parts and developmental periods. The overall mass fractions of IAA and ABA were significantly higher than those of ZR and GA, driven by differences among bamboo parts and developmental periods. The abundance of each endogenous hormone varied among bamboo parts and developmental periods. During bamboo shooting, ABA had the highest mass fraction in all bamboo parts sampled, followed by IAA, GA, and ZR. Among bamboo parts, rhizomes had more IAA, ZR, and GA than the other parts, but significantly less ABA. Winter shoots had higher ZR: IAA and GA: IAA ratios than rhizomes and maternal bamboo organs. During shoot growth, ABA was the most abundant hormone in rhizomes and maternal bamboo organs, followed by IAA, ZR, and GA. In contrast, IAA was the most abundant hormone in spring shoots, followed by ABA, ZR, and GA. Maternal bamboo organs had a significantly higher ZR: GA ratio, and significantly lower IAA: ABA, ZR: ABA, and GA: ABA ratios than rhizomes. Spring shoots had significantly higher IAA: ABA, ZR: ABA, and GA: ABA ratios than rhizomes and maternal bamboo organs; significantly higher ZR mass fractions, and ZR: GA and ZR: IAA ratios and significantly lower ABA mass fractions than rhizomes; and significantly higher GA: IAA ratio than maternal bamboo organs. During new-bamboo formation, ABA was the most abundant hormone in rhizomes, winter shoots, and maternal bamboo organs, followed by IAA, ZR, and GA. Maternal bamboo organs had significantly lower IAA mass fractions and significantly higher ABA mass fractions than rhizomes and new bamboo tissue. IAA and ABA abundances exhibited an inverse relationship in rhizomes and maternal bamboo organs. GA: ABA and GA: IAA ratios decreased gradually and other hormone ratios exhibited parabolic trends over the bamboo-shooting period, with the highest ratios observed in new bamboo tissues. Overall, the coordination or antagonism among endogenous hormones plays a key regulatory role in bamboo shoot growth. The formation of thick walls in P. edulis 'Pachyloen', one of its major traits, may be partially attributed to the relatively high IAA and ZR and low GA mass fractions.

Phytohormone profile in Lactuca sativa and Brassica oleracea plants grown under Zn deficiency.

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.

[Determination of 6 kinds of plant growth regulator in bean sprout by ultra high performance liquid chromatography-tandem mass spectrometry].

OBJECTIVE: A method for the simultaneous determination of 6 plant growth regulator (PGR) residues in bean sprout was developed by ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). 6-Benzylaminopurine, isopentennyladenine, 4-chlorophenoxyacetic acid, 4-fluorophenoxyacetic acid, indole-3- acetic acid and indole-3-butyric acid were concerned. METHODS: Bean sprout samples were extracted by acetonitrile and QuEChERS extraction kit, purified by C18 powers. After centrifugation, the sample liquids was diluted 10 times by ultrapure water. The chromatographic analysis was carried out on an waters acquity UPLC BEH C18 column( 100 mm x 2.1 mm, 1.7 microm). The analyzer confirmed and quantified by mass spectrum of triple quadrupole in the multiple reaction monitoring (MRM) mode and quantified by matrix-matched external standard method. RESULTS: The calibration curves showed good linearity in each range with correlation coefficients greater than 0.998. 3 levels spiked recoveries were carried out using blank bean sprout extraction as substrate, the recoveries ranged from 84.2% to 107.5%, the relative standard deviations (RSDs) ranged from 3.08% to 12.71%. The qualitative limits of detections (S/N = 3) were 0.03-3.0 microg/kg and the quantitative limits(S/N = 10) were 0.1-10.0 microg/kg for the 6 PGRs. CONSLUSION: The method is simple and easy to operate, with less organic reagent, high sensitivity and good stability. It is suitable for the detection of 6 kinds of plant growth regulators in bean sprouts.

Simple and rapid determination of N(6)-(Δ(2)-isopentenyl)adenine, zeatin, and dihydrozeatin in plants using on-line cleanup liquid chromatography coupled with hybrid quadrupole-Orbitrap high-resolution mass spectrometry.

A simple and rapid method was developed for the determination of three free cytokinins, namely, N(6)-(Δ(2)-isopentenyl)adenine, zeatin, and dihydrozeatin, in plants using TurboFlow on-line cleanup liquid chromatography combined with hybrid quadrupole-Orbitrap high-resolution mass spectrometry. The samples were extracted using acetonitrile, and then the extract was purified on a C18-p column, in which the sample matrix was removed and the analytes were retained. Subsequently, the analytes were eluted from the extraction column onto the analytical column (Hypersil Gold C18 column) prior to chromatographic separation and hybrid Q-Orbitrap detection using the targeted-MS(2) scan mode. The linearity was satisfactory with a correlation coefficient of >0.999 at concentrations ranging from 5-5000 pg/mL. The limits of quantification for the analytes ranged from 4.2-5.2 pg/mL. The intra- and inter-day average recoveries of analytes fortified at three levels ranged from 85.4-108.2%, and the intra- and inter-day relative standard deviations ranged from 4.04-8.57%. The method was successfully applied for the determination of free cytokinins in different tissue samples of Oryza sativa and Arabidopsis thaliana.

A highly selective biosensor with nanomolar sensitivity based on cytokinin dehydrogenase.

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.

Simultaneous quantification of phytohormones in fermentation extracts of Botryodiplodia theobromae by liquid chromatography-electrospray tandem mass spectrometry.

Fermentation broth and biomass from three strains of Botryodiplodia theobromae were characterized by high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) method, in order to quantify different phytohormones and to identify amino acid conjugates of jasmonic acid (JA) present in fermentation broths. A liquid-liquid extraction with ethyl acetate was used as sample preparation. The separation was carried out on a C18 reversed-phase HPLC column followed by analysis via ESI-MS/MS. The multiple reaction monitoring mode was used for quantitative measurement. For the first time, indole-3-acetic acid, indole-3-propionic acid, indole-3-butyric acid and JA were identified and quantified in the ethyl acetate extracts from the biomass, after the separation of mycelium from supernatant. The fermentation broths showed significantly higher levels of JA in relation to the other phytohormones. This is the first report of the presence of gibberellic acid, abscisic acid, salicylic acid and the cytokinins zeatin, and zeatin riboside in fermentation broths of Botryodiplodia sp. The presence of JA-serine and JA-threonine conjugates in fermentation broth was confirmed using HPLC-ESI tandem mass spectrometry in negative ionization mode, while the occurrence of JA-glycine and JA-isoleucine conjugates was evidenced with the same technique but with positive ionization. The results demonstrated that the used HPLC-ESI-MS/MS method was effective for analysing phytohormones in fermentation samples.

Effects of nitric oxide treatment on the cell wall softening related enzymes and several hormones of papaya fruit during storage.

Papaya fruits (Carica papaya L. cv 'Sui you 2') harvested with < 5% yellow surface at the blossom end were fumigated with 60 microL/L of nitric oxide for 3 h and then stored at 20 degrees C with 85% relative humility for 20 days. The effects of nitric oxide treatment on ethylene production rate, the activities of cell wall softening related enzymes including polygalacturonase, pectin methyl esterase, pectate lyase and cellulase and the levels of hormones including indole acetic acid, abscisic acid, gibberellin and zeatin riboside were examined. The results showed that papaya fruits treated with nitric oxide had a significantly lower rate of ethylene production and a lesser loss of firmness during storage. A decrease in polygalacturonase, pectin methyl esterase, pectate lyase and cellulase activities was observed in nitric oxide treated fruit. In addition, the contents of indole acetic acid, abscisic acid and zeatin riboside were reduced in nitric oxide treated fruit, but no significant reduction in the level of gibberellin was found. These results indicate that nitric oxide treatment can effectively delay the softening and ripening of papaya fruit, likely via the regulation of cell wall softening related enzymes and certain hormones.

[Effects of local induction of ipt-gene in roots on cytokinins content in leaf cells tobacco plants].

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.

The CDK5 repressor CDK5RAP1 is a methylthiotransferase acting on nuclear and mitochondrial RNA.

The unusual cyclin-dependent protein kinase 5 (CDK5) was discovered based on its sequence homology to cell cycle regulating CDKs. CDK5 was found to be active in brain tissues, where it is not involved in cell cycle regulation but in the regulation of neuronal cell differentiation and neurocytoskeleton dynamics. An aberrant regulation of CDK5 leads to the development of various neurodegenerative diseases including Alzheimer's disease. Although CDK5 is not regulated by cyclins, its activity does depend on the association with a protein activator and the presence or absence of further inhibitory factors. Recently, CDK5RAP1 was discovered to inhibit the active CDK5 kinase. Here, we show that CDK5RAP1 is a radical SAM enzyme, which postsynthetically converts the RNA modification N6-isopentenyladenosine (i(6)A) into 2-methylthio-N6-isopentenyladenosine (ms(2)i(6)A). This conversion is surprisingly not limited to mitochondrial tRNA, where the modification was known to exist. Instead, CDK5RAP1 introduces the modification also into nuclear RNA species establishing a link between postsynthetic kinase-based protein modification and postsynthetic RNA modification.

A liquid chromatography tandem mass spectrometry method for simultaneous determination of acid/alkaline phytohormones in grapes.

A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneous determination of five acid/alkaline phytohormones, i.e., indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthylacetic acid (NAA), gibberellic acid (GA(3)) and isopentenyladenine (2IP), in grapes was developed. After optimization, the samples were extracted with methanol containing 1% formic acid and purified by Oasis HLB SPE cartridges. The analytes were separated on a Thermo Hypersil Gold column (100 mm×2.1 mm, 3.0 µm) with water and acetonitrile, then determined with Thermo tandem quadrupole mass spectrometer operating in negative electro-spray ionization using selected reaction monitoring (SRM) mode. The established method was further validated by determining the linearity (R² ≥ 0.9990), average recovery (82.5-105.4%), sensitivity (0.05-1.00 ng mL⁻¹), precision (RSD ≤1 3.0%) and stability (RSD ≥ 82.0%). Finally, the application of the approach proposed to thirty grape samples convinced its desirable performance for rapid analysis of multiclass phytohormones, supporting its sufficient capability for multiresidue analyses or other analytical system targeting phytohormones in agriculture field.

Elevated carbon dioxide and/or ozone concentrations induce hormonal changes in Pinus tabulaeformis.

We investigated endogenous plant hormones and needle growth in Pinus tabulaeformis plants grown in open-top chambers and exposed to ambient or elevated concentrations of carbon dioxide (CO(2)) and/or ozone (O(3)). Exposure to elevated CO(2) for 100 days significantly increased the change in fresh needle weight, indole-3-acetic acid (IAA), isopentenyl-adenosine (iPA), and dihydrozeatin riboside (DHZR) content. Abscisic acid (ABA) content decreased, and no effect was observed on zeatin riboside (ZR) content or changes in needle dry weight. The ratios of IAA/ABA and total cytokinins (CKs)/ABA [Formula: see text] were increased. Elevated O(3) significantly decreased IAA and ZR, and decreased the ratios of IAA/ABA and CKs/ABA. Ozone treatment increased ABA content but did not change iPA or DHZR content or change fresh or dry needle weights. The combination treatment significantly increased ABA content and the IAA/ABA ratio but decreased the total CKs/ABA ratio and had no effect on CKs or IAA content or change in fresh and dry needle weights. The results indicate that elevated CO(2) ameliorated the effects of elevated O(3) on tree growth.

1H, 13C and 15N NMR spectral assignments of adenosine derivatives with different amino substituents at C6-position.

The complete (1)H, (13)C and (15)N NMR signals assignment of adenosine derivatives differently substituted at C(6)-position was achieved using one- and two-dimensional experiments (gs-COSY, gs-NOESY, gs-HSQC and gs-HMBC).

Influence of plant maturity, shoot reproduction and sex on vegetative growth in the dioecious plant Urtica dioica.

BACKGROUND AND AIMS: Stinging nettle (Urtica dioica) is a herbaceous, dioecious perennial that is widely distributed around the world, reproduces both sexually and asexually, and is characterized by rapid growth. This work was aimed at evaluating the effects of plant maturity, shoot reproduction and sex on the growth of leaves and shoots. METHODS: Growth rates of apical shoots, together with foliar levels of phytohormones (cytokinins, auxins, absicisic acid, jasmonic acid and salicylic acid) and other indicators of leaf physiology (water contents, photosynthetic pigments, alpha-tocopherol and F(v)/F(m) ratios) were measured in juvenile and mature plants, with a distinction made between reproductive and non-reproductive shoots in both males and females. Vegetative growth rates were not only evaluated in field-grown plants, but also in cuttings obtained from these plants. All measurements were performed during an active vegetative growth phase in autumn, a few months after mature plants reproduced during spring and summer. KEY RESULTS: Vegetative growth rates in mature plants were drastically reduced compared with juvenile ones (48 % and 78 % for number of leaves and leaf biomass produced per day, respectively), which was associated with a loss of photosynthetic pigments (up to 24 % and 48 % for chlorophylls and carotenoids, respectively) and increases of alpha-tocopherol (up to 2.7-fold), while endogenous levels of phytohormones did not differ between mature and juvenile plants. Reductions in vegetative growth were particularly evident in reproductive shoots of mature plants, and occurred similarly in both males and females. CONCLUSIONS: It is concluded that (a) plant maturity reduces vegetative growth in U. dioica, (b) effects of plant maturity are evident both in reproductive and non-reproductive shoots, but particularly in the former, and (c) these changes occur similarly in both male and female plants.

Changes of zeatin riboside content in rice plants due to infestation by Nilaparvata lugens (Homoptera: Delphacidae).

The effect of Nilaparvata lugens (Stål) (Homoptera: Delphacidae), infestation on the content of zeatin ribosides (ZR) in rice plants was investigated with enzyme-linked immunosorbent assay. Hydroponics experiments were conducted on 'Zhendao 2' rice, in which plants were subjected to N. lugens infestation at three nonhopperburn-causing densities (15, 30, and 60 nymphs per hill) for 2, 4, 6, and 8 d and at one hopperburn-causing density (240 nymphs per hill) for 2, 4, and 6 d, respectively. When rice plants were infested at the nonhopperburn-causing densities, ZR content in leaves varied significantly with the infestation density. Compared with the control plants, ZR content in rice leaves decreased significantly after infestation by 60 nymphs per hill for 2 d, but it tended to increase due to prolonged infestation at all the nonhopperburn-causing densities. In contrast, ZR content in rice roots significantly reduced after the plants being infested at the density of 15 nymphs for 2 d and at all densities for prolonged duration, except for the plants infested by 60 nymphs for 6 and 8 d, in which the ZR content increased or did not change significantly. However, infestation at the hopperburn-causing density caused significant reduction in ZR content in rice roots, regardless of infestation duration, and in rice leaves from the plants subjected to 2-d infestation. These results are discussed in relation to the possible physiological reaction of rice plants to N. lugens infestation and the resultant severe damage or hopperburn.

Investigation of cytokinin-deficient phenotypes in Arabidopsis by ectopic expression of orchid DSCKX1.

The plant hormone cytokinin plays a major role in regulating plant growth and development. Here we generated cytokinin-reduction Arabidopsis plants by overexpressing a heterologous cytokinin oxidase gene DSCKX1 from Dendrobium orchid. These transgenic plants exhibited reduced biomass, rapid root growth, decreased ability to form roots in vitro, and reduced response to cytokinin in growing calli and roots. Furthermore, the expression of KNAT1, STM, and CycD3 genes was significantly reduced in the transgenic plants, suggesting that cytokinin may function to control the cell cycles and shoot/root development via regulation of these genes.

Amperometric immunosensor based on polypyrrole/poly(m-pheylenediamine) multilayer on glassy carbon electrode for cytokinin N6-(Delta2-isopentenyl) adenosine assay.

A novel immunosensor based on a multilayer-coated glassy carbon electrode was designed to determine isopentenyl adenosine (iPA) in plants. The multilayer consists of polypyrrole and poly(m-phenylenediamine) with K4Fe(CN)6 and horseradish peroxidase (HRP) entrapped during electropolymerization. The ferrocyanide doped in polypyrrole functions as the mediator. The glucose oxidase bound on the immunosensor by the competitive immunoreaction involving iPA catalyzed the oxidation of the added glucose with the formation of H2O2, which is in turn reduced in the presence of HRP entrapped in poly(m-phenylenediamine). The current of the oxidized production of ferrocyanide reduced at -50 mV is inversely proportional to the concentration of iPA in the competitive immunoreaction. This immunosensor is able to be used about 40 times; after that its surface can be regenerated for a new immunosensor assembly by washing with 0.1M citrate-phosphate buffer (pH 4.6). The percentage of current response reduction (CR%) (y) is linearly related to the logarithm of the concentration of iPA (x) in the 5-300 microg/ml range, with a regression equation of the form y = 42.13x - 27.79 and a correlation coefficient of 0.9861. Five hybrid rice grain samples were analyzed with results in satisfactory agreement to those obtained by high-performance liquid chromatography.

Charcoal affects early development and hormonal concentrations of somatic embryos of hybrid larch.

Embryogenic tissue of hybrid larch (Larix x marschlinsii Coaz) was multiplied on Medium M (modified MSG medium supplemented with the plant growth regulators (PGRs) 2,4-dichlorophenoxyacetic acid (2,4-D; 9 microM) and N-6-benzyladenine (2.25 microM)). After 1 week, cultures were transferred to either MSG lacking PGRs (Medium C-) or MSG lacking PGRs but supplemented with 1% activated charcoal (Medium C+). Embryos were sampled after 1 week on Medium M, C- or C+. Embryos were analyzed by ELISA for abscisic acid (ABA), abscisic acid-glucose ester, 2,4-D, indole-3-acetic acid (IAA), indole-3-aspartate (IAAsp), zeatin (Z), zeatin riboside (ZR), isopentenyladenine (iP) and isopentenyladenosine (iPA). Transfer of embryos to Medium C+ reduced the embryo concentrations of 2,4-D and iPA, but resulted in elevated concentrations of IAA, IAAsp, ABA, Z, ZR and iP. Charcoal reduced 2,4-D concentrations of embryos by an order of magnitude greater than PGR-free medium alone. Charcoal affected embryo concentrations of five of the eight PGRs quantified. Use of either C+ or C- medium as part of the maturation protocols also affected germination and plantlet establishment of the embryos. A 1-week treatment on Medium C+ positively influenced plantlet establishment and generally reduced variability during both germination and plantlet establishment.

[A new micromethod for differential quantitative assay of zeatin and zeatin riboside]. / Novyi mikrometod differentsial'nogo kolichestvennogo opredeleniia zeatina i zeatinribozida.

A new method is proposed for differential quantitative assay of two major endogenous cytokinin forms. It is based on determination of two effective parameters-concentrations of zeatin and zeatin riboside--with the use of appropriate antigens as standards. The method can be used for determining cytokinins in small samples of plant tissues without extract fractionation. This study pioneers in quantitation of changes in the hormonal status of ovules and ovaries of Triticum aestivum L. at early stages of embryogeny. A gradual increase in the content of the active and storage forms of the hormones from the ovary to the ovule was revealed.