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Callus cultures of the Iranian medicinal plant Salvia atropatana were initiated from three-week-old seedlings on Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA) and various cytokinins. Although all tested hormonal variants of the medium and explant enabled callus induction, the most promising growth was noted for N -(2-chloro-4-pyridyl)- N '-phenylurea (CPPU)-induced calli. Three lines obtained on this medium (cotyledon line-CL, hypocotyl line-HL, and root line-RL) were preselected for further studies. Phenolic compounds in the callus tissues were identified using UPLC-MS (ultra-performance liquid chromatography-mass spectrometry) and quantified with HPLC (high-performance liquid chromatography). All lines exhibited intensive growth and contained twelve phenolic acid derivatives, with rosmarinic acid predominating. The cotyledon-derived callus line displayed the highest growth index values and polyphenol content; this was exposed to different light-emitting diodes (LED) for improving biomass accumulation and secondary metabolite yield. Under LED treatments, all callus lines exhibited enhanced RA and total phenolic content compared to fluorescent light, with the highest levels observed for white (48.5-50.2 mg/g dry weight) and blue (51.4-53.9 mg/g dry weight) LEDs. The selected callus demonstrated strong antioxidant potential in vitro based on the 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and ferric reducing antioxidant power (FRAP) tests. Our findings confirm that the S. atropatana callus system is suitable for enhanced rosmarinic acid production; the selected optimized culture provide high-quality plant-derived products.

In vitro cell culturing witnessed its applications in scientific research and industrial activities. Attempts to shorten the doubling time of cultured cells have never ceased. In plants, auxin is applied to promote plant growth, the synthetic derivative 1-Naphthaleneacetic acid (NAA) is a good example. Despite the auxin's naturally occurring receptors are not present in mammalian cells, studies suggested they may affect cell culturing. Yet the effects and mechanisms are still unclear. Here, an up to 2-fold increase in the yield of in vitro cultured human cells is observed. Different types of human cell lines and primary cells are tested and found that NAA is effective in all the cells tested. The PI staining followed by FACS suggested that NAA do not affect the cell cycling. Apoptosis-specific dye staining analysis implicated that NAA rescued cell death. Further bulk RNA sequencing is done and it is identified that the lipid metabolism-engaging and anti-apoptosis gene, ANGPTL4, is enhanced in expression upon NAA treatment. Studies on ANGPTL4 knockout cells indicated that ANGPTL4 is required for NAA-mediated response. Thus, the data identified a beneficial role of NAA in human cell culturing and highlighted its potency in in vitro cell culturing., (© 2024 Wiley-VCH GmbH.)

Coupling microalgae cultivation with wastewater treatment is a promising environmentally sustainable development strategy. However, toxics such as Bisphenol A (BPA) in wastewater damage microalgae cells and reduces bioresources production. Phytohormone regulation has the potential to solve this issue. However, phytohormone research is still in its infancy. In this work, 0.2 μM naphthyl acetic acid (NAA) significantly enhanced Chlorella vulgaris BPA detoxification by 127.3% and Chlorella biomass production by 46.4%. NAA helps Chlorella convert bisphenol A into small non-toxic intermediates by enhancing the expression of associated enzymes. Simultaneously, NAA promoted carbon fixation and photosynthetic metabolism. Activation of the mitogen-activated protein kinase (MAPK) pathway strengthened the downstream antioxidant system while improving photosynthesis and intracellular starch and lipid synthesis. Carbohydrates, pigment, and lipid production was significantly enhanced by 20.0%, 46.9%, and 21.8%, respectively. A new insight is provided into how phytohormones may increase microalgae in wastewater's bioresource transformation and toxicity resistance., 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 © 2022. Published by Elsevier Ltd.)

Nabumetone (NAB) is a non-steroidal anti-inflammatory drug used clinically, and its biotransformation includes the major active metabolite 6-methoxy-2-naphthylacetic acid (6-MNA). One of the key intermediates between NAB and 6-MNA may be 3-hydroxy nabumetone (3-OH-NAB). The aim of the present study was to investigate the role of flavin-containing monooxygenase (FMO) isoform 5 in the formation of 6-MNA from 3-OH-NAB. To elucidate the biotransformation of 3-OH-NAB to 6-MNA, an authentic standard of 3-OH-NAB was synthesised and used as a substrate in an incubation with human liver samples or recombinant enzymes. The formation of 3-OH-NAB was observed after the incubation of NAB with various cytochrome P450 (CYP) isoforms. However, 6-MNA itself was rarely detected from NAB and 3-OH-NAB. Further experiments revealed a 6-MNA peak derived from 3-OH-NAB in human hepatocytes. 6-MNA was also detected in the extract obtained from 3-OH-NAB by a combined incubation of recombinant human FMO5 and human liver S9. We herein demonstrated that the reaction involves carbon-carbon cleavage catalyzed by the Baeyer-Villiger oxidation (BVO) of a carbonyl compound, the BVO substrate, such as a ketol, by FMO5. Further in vitro inhibition experiments showed that multiple non-CYP enzymes are involved in the formation of 6-MNA from 3-OH-NAB.

The abscisic acid (ABA) increase and auxin decline are both indicators of ripening initiation in grape berry, and norisoprenoid accumulation also starts at around the onset of ripening. However, the relationship between ABA, auxin, and norisoprenoids remains largely unknown, especially at the transcriptome level. To investigate the transcriptional and posttranscriptional regulation of the ABA and synthetic auxin 1-naphthaleneacetic acid (NAA) on norisoprenoid production, we performed time-series GC-MS and RNA-seq analyses on Vitis vinifera L. cv. Cabernet Sauvignon grape berries from pre-veraison to ripening. Higher levels of free norisoprenoids were found in ABA-treated mature berries in two consecutive seasons, and both free and total norisoprenoids were significantly increased by NAA in one season. The expression pattern of known norisoprenoid-associated genes in all samples and the up-regulation of specific alternative splicing isoforms of VviDXS and VviCRTISO in NAA-treated berries were predicted to contribute to the norisoprenoid accumulation in ABA and NAA-treated berries. Combined weighted gene co-expression network analysis (WGCNA) and DNA affinity purification sequencing (DAP-seq) analysis suggested that VviGATA26, and the previously identified switch genes of myb RADIALIS (VIT_207s0005g02730) and MAD-box (VIT_213s0158g00100) could be potential regulators of norisoprenoid accumulation. The positive effects of ABA on free norisoprenoids and NAA on total norisoprenoid accumulation were revealed in the commercially ripening berries. Since the endogenous ABA and auxin are sensitive to environmental factors, this finding provides new insights to develop viticultural practices for managing norisoprenoids in vineyards in response to changing climates.

Endophytic fungi can be beneficial to plant growth. However, the molecular mechanisms underlying colonization of Acremonium spp. remain unclear. In this study, a novel endophytic Acremonium strain was isolated from the buds of Panax notoginseng and named Acremonium sp. D212. The Acremonium sp. D212 could colonize the roots of P. notoginseng, enhance the resistance of P. notoginseng to root rot disease, and promote root growth and saponin biosynthesis in P. notoginseng. Acremonium sp. D212 could secrete indole-3-acetic acid (IAA) and jasmonic acid (JA), and inoculation with the fungus increased the endogenous levels of IAA and JA in P. notoginseng. Colonization of the Acremonium sp. D212 in the roots of the rice line Nipponbare was dependent on the concentration of methyl jasmonate (MeJA) (2-15 μmol/L) and 1-naphthalenacetic acid (NAA) (10-20 μmol/L). Moreover, the roots of the JA signaling-defective coi1-18 mutant were colonized by Acremonium sp. D212 to a lesser degree than those of the wild-type Nipponbare and miR393b-overexpressing lines, and the colonization was rescued by MeJA but not by NAA. It suggests that the cross-talk between JA signaling and the auxin biosynthetic pathway plays a crucial role in the colonization of Acremonium sp. D212 in host plants., (© 2020 Institute of Botany, Chinese Academy of Sciences.)

The pathway for the transformation of the prodrug nabumetone, 4-(6-methoxynaphthalen-2-yl)butan-2-one, to the active metabolite 6-methoxy-2-naphthylacetic acid (6-MNA), a potent cyclooxygenase-2 inhibitor, has not yet been clarified in humans.To confirm the activation pathway, authentic standards of the nabumetone intermediates, 2-(6-methoxynaphthalen-2-yl)ethyl acetate (6-MNEA), 2-(6-methoxynaphthalen-2-yl)ethan-1-ol (6-MNE-ol) and 2-(6-methoxynaphthalen-2-yl)acetaldehyde (6-MN-CHO) were synthesized. High performance liquid-chromatography and gas chromatography-mass spectrometry on nabumetone oxidation revealed the generation of three metabolites.The formation of 6-MNA after a 60-min incubation of nabumetone was detected and 6-MNE-ol, an alcohol-related intermediate, was also generated by in cryopreserved hepatocytes. However, 6-MNA was below detection limit, but 4-(6-methoxynaphthalen-2-yl)butan-2-ol (MNBO) and 4-(6-hydroxynaphthalen-2-yl)butan-2-one (M3) peak were found in both the microsomes and S9 extracts with any cofactors.Nabumetone has recently been proposed as a typical substrate of flavin-containing monooxygenase isoform 5 (FMO5) and was shown to be efficiently oxidized in vitro to 6-MNEA. 6-MNA was detected in the extract obtained from a combined incubation of recombinant FMO5 and S9 fractions.The specificity of FMO5 towards catalyzing this Baeyer-Villiger oxidation (BVO) was demonstrated by the inhibition of the BVO substrate, 4-methoxyphenylacetone. Further in vitro inhibition studies demonstrated that multiple non-cytochrome P450 enzymes are involved in the formation of 6-MNA.

The roles of C-terminal Eps15 homology domain (EHD) proteins in clathrin-mediated endocytosis in plants are poorly understood. Here, we isolated a maize ( Zea mays ) mutant, designated ehd1 , which showed defects in kernel development and vegetative growth. Positional cloning and transgenic analysis revealed that ehd1 encodes an EHD protein. Internalization of the endocytic tracer FM4-64 was substantially reduced in the ehd1 mutant and ZmEHD1 knockout mutants. We further demonstrated that ZmEHD1 and the ZmAP2 σ subunit physically interact at the plasma membrane. Auxin distribution and ZmPIN1a-YFP localization were altered in the ehd1 mutant. Kernel indole-3-acetic acid levels were substantially lower in the ehd1 mutant than in wild-type maize. Exogenous application of 1-naphthaleneacetic acid, but not GA 3 or 2-naphthaleneacetic acid, rescued the seed germination and seedling emergency phenotypic defects of ehd1 mutants. Taken together, these results indicate that ZmEHD1 regulates auxin homeostasis by mediating clathrin-mediated endocytosis through its interaction with the ZmAP2 σ subunit, which is crucial for kernel development and vegetative growth of maize., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

The novel methods for efficient plant regeneration via direct somatic embryogenesis (SE) and SE-mediated transformation system under high concentration of NAA in Ranunculus sceleratus were established. On MS media containing a high concentration of NAA (10.0 mg/L) in the dark, all inoculated explants (root, stem and leaf) formed somatic embryos at high frequencies, respectively, 66.03, 126.47 and 213.63 embryoids per explant, and 100% of the embryoids developed into plantlets on 1/2 MS rooting media. Morphological and histological analyses revealed that SE in R. sceleratus followed a classical pattern. All inoculated explants can be used as receptors for genetic transformation in R. sceleratus, through direct SE-mediated method after Agrobacterium infection. RcLEC1-B, as a marker gene, changed the number and morphology of flower organs and the development of cuticle in R. sceleratus, which indicated that the efficient transgenic system of R. sceleratus was established. To our knowledge, this is the first observation that both direct SE and transgenic transformation system, via induction of a single plant growth regulator, have been successfully constructed in R. sceleratus.

1. The underlying microbial metabolic activity toward xenobiotics is among the least explored factors contributing to the inter-individual variability in drug response. 2. Here, we analyzed the effect of microbiota on a non-steroidal anti-inflammatory drug nabumetone. 3. First, we cultivated the drug with the selected gut commensal and probiotic bacteria under both aerobic and anaerobic conditions and analyzed its metabolites by high-performance liquid chromatography (HPLC) with UV detection. To analyze the effect of microbiota on nabumetone pharmacokinetics in vivo , we administered a single oral dose of nabumetone to rodents with intentionally altered gut microbiome - either rats treated for three days with the antibiotic imipenem or to germ-free mice. Plasma levels of its main active metabolite 6 methoxy-2-naphthylacetic acid (6-MNA) were analyzed at pre-specified time intervals using HPLC with UV/fluorescence detection. 4. We found that nabumetone is metabolized by bacteria to its non-active metabolites and that this effect is stronger under anaerobic conditions. Although in vivo , none of the pharmacokinetic parameters of 6-MNA was significantly altered, there was a clear trend towards an increase of the AUC, Cmax and t 1/2 in rats with reduced microbiota and germ-free mice.

1-Naphthaleneacetic acid (NAA) has long been used to regulate strawberry growth. However, its regulatory mechanisms are unclear. Here, a nuclear magnetic resonance (NMR)-based metabolomics approach was utilized to capture differential metabolites, then matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and transcriptomics as assisted methods to validate the significant findings of metabolomics. The metabolomics results suggested that NAA regulated strawberry growth via multiple metabolic pathways, and different NAA application times also influenced these regulatory effects. We also found an interesting phenomenon that citric acid had completely opposite changes when NAA was sprayed at two different ripening stages of the strawberries. Furthermore, MALDI-TOF MS validated the changes of citric acid and transcriptomics identified the related genes. The study demonstrated that the novel strategy of "metabolomics capture-MALDI-TOF MS and transcriptomics assisted validation" could offer a fresh insight for understanding the mechanism of the plant growth regulator in strawberry maturation.

Short peptide-based hydrogels have attracted extensive research interests in drug delivery because of their responsive properties. So far, most drug molecules have been conjugated with short peptides via an amide bond, restricting the release of the native drug molecules. In this study, we demonstrated the effectiveness of an auxin-based hydrogelator linked by a hydrolysable ester bond. Hydrogel I, formed by the gelator (NAA-G'FFY) linked with an ester bond, was able to release 1-naphthaleneacetic acid (NAA), whereas hydrogel II, formed by the gelator without an ester bond (NAA-GFFY), was not. By mixing NAA-G'FFY with Fmoc-GFFY to form a two-component hydrogel, the spatial and temporal release of NAA was achieved, promoting on-site auxin responses including primary root elongation and lateral root formation in the model plant Arabidopsis thaliana. The strategy of using a hydrolysable ester bond to connect drug molecules and self-assembling peptides could lead to the development of supramolecular hydrogels with more controllable drug release profiles.

Brewery effluent (BE) as an appreciable and sustainable resource presented new possibilities in low-cost algal biomass production, whereas the relatively low essential macronutrients hindered extensive applications as growth medium for microalgae cultivation. The objective of this study was to investigate the feasibility of an integrated treatment strategy initiated by BE coupling phytohormones in augmenting biomass and lipid accumulation in Coccomyxa subellipsoidea. Results revealed that BE coupling synthetic 1-naphthaleneacetic acid (NAA) accomplished the favorable lipid productivity of 481.76 mg/L/days, representing 6.80- to 9.71-fold more than that of single BE as well as standard Basal media. BE coupling NAA feeding also heightened the proportions of C16-C18 fatty acids (over 96%) and mono-unsaturated C18:1 (approximate 45%) which were prone to high-quality biofuels-making. Such profound lipids accumulation might be attributable to that BE coupling NAA treatment drove most of metabolic flux (i.e. acetyl-CoA) derived from TCA cycle and glycolysis flowing into lipid accumulation pathway. Concurrently, the complete removal of total nitrogen and total phosphorus by C. subellipsoidea with assistance of NAA were easily complied with the permissible dischargeable limits for BE. These present results strongly demonstrated that BE coupling NAA was a potential feeding strategy in boosting algal lipid productivity and further provided great possibilities in linking affordable algal biomass production with high-efficient biological contaminants removal.

Key Message: A fresh look at the roles of auxin, ethylene, and polar auxin transport during the plant root growth response to warmer ambient temperature (AT). The ambient temperature (AT) affects plant growth and development. Plants can sense changes in the AT, but how this change is transduced into a plant root growth response is still relatively unclear. Here, we found that the Arabidopsis ckrc1-1 mutant is sensitive to higher AT. At 27 °C, the ckrc1-1 root length is significantly shortened and the root gravity defect is enhanced, changes that can be restored with addition of 1-naphthaleneacetic acid, but not indole-3-acetic acid (IAA). AUX1, PIN1, and PIN2 are involved in the ckrc1-1 root gravity response under increased AT. Furthermore, CKRC1-dependent auxin biosynthesis was critical for maintaining PIN1, PIN2, and AUX1 expression at elevated temperatures. Ethylene was also involved in this regulation through the ETR1 pathway. Higher AT can promote CKRC1-dependent auxin biosynthesis by enhancing ETR1-mediated ethylene signaling. Our research suggested that the interaction between auxin and ethylene and that the interaction-mediated polar auxin transport play important roles during the plant root growth response to higher AT.

Strawberry fruit contain the allergenic Fra a proteins, members of the pathogenesis-related 10 protein family that causes oral allergic syndrome symptoms. Fra a proteins are involved in the flavonoid biosynthesis pathway, which might be important for color development in fruits. Auxin is an important plant hormone in strawberry fruit that controls fruit fleshiness and ripening. In this study, we treated strawberry fruits with exogenous auxin or auxin inhibitors at pre- and post-harvest stages, and analyzed Fra a transcriptional and translational expression levels during fruit development by real-time PCR and immunoblotting. Pre-harvest treatment with 1-naphthaleneacetic acid (NAA) alone did not affect Fra a expression, but applied in conjunction with achene removal NAA promoted fruit pigmentation and Fra a protein accumulation. The response was developmental stage-specific: Fra a 1 was highly expressed in immature fruit, whereas Fra a 2 was expressed in young to ripe fruit. In post-harvest treatments, auxin did not contribute to Fra a induction. Auxin inhibitors delayed fruit ripening; as a result, they seemed to influence Fra a 1 expression. Thus, Fra a expression was not directly regulated by auxin, but might be associated with the ripening process and/or external factors in a paralog-specific manner., Competing Interests: The authors declare no conflict of interest.

Malus hupehensis is an excellent Malus rootstock species, known for its strong adverse-resistance and apomixes. In the present study, stem cuttings of M. hupehensis were treated with three types of exogenous hormones, including indole acetic acid (IAA), naphthalene acetic acid (NAA), or green growth regulator (GGR). The effects and mechanisms of exogenous hormone treatment and antioxidant enzyme activity on adventitious root formation were investigated. The results showed that the apparent morphology of the adventitious root had four stages, including root pre-emergence stage (S0), early stage of root formation (S1), massive root formation stage (S2), and later stage of root formation (S3). The suitable concentrations of the three exogenous hormones, IAA, NAA and GGR, were 100 mg·L-1, 300 mg·L-1, and 300 mg·L-1, respectively. They shortened the rooting time by 25-47.4% and increased the rooting percentages of cuttings by 0.9-1.3 times, compared with that in the control. The dispersion in S0 stage was 3.6 times of that in the S1 stage after exogenous hormone application. The earlier the third critical point (P3) appeared, the shorter the rooting time and the greater the rooting percentage of the cuttings. During rhizogenesis, the activities of three antioxidant enzymes (POD, SOD, and PPO) showed an A-shaped trend. However, peak values of enzyme activity appeared at different points, which were 9 d before the P3, P3, and the fourth critical point (P4), respectively. Exogenous hormone treatment reduced the time to reach the peak value by 18 days, although the peak values of the enzymatic activities did not significantly changed. Our results suggested that exogenous hormone treatment mainly acted during the root pre-emergence stage, accelerated the synthesis of antioxidant enzymes, reduced the rooting time, and consequently promoted root formation. The three kinds of antioxidant enzymes acted on different stages of rooting.

In the current study, we evaluated the effect of α-naphthaleneacetic acid (NAA) individually or in combination with different cytokinins (CKs) including benzyladenine (BA), meta-topolin (mT) and isopentenyladenine (iP) on organogenesis, auxin and CK content in Eucomis autumnalis subspecies autumnalis (EA) and Eucomis zambesiaca (EZ). These species were used as model plants due to their ornamental and medicinal properties. Three leaf explants were inoculated in screw-cap jars containing 30mL Murashige and Skoog (MS) media supplemented with 5μM NAA alone or in combination with 5μM CK (BA, mT or iP). After 10 weeks (EA) or 15 weeks (EZ), parameters including shoot and root growth as well as plant fresh weight were recorded. For analysis of auxin and CK content, whole plantlets were harvested, pooled and freeze-dried for the different treatments. In both species, shoot and root proliferation as well as plant biomass were generally higher when NAA was combined with the individual CK than in NAA or CK treatment. The highest concentration of indole-3-acetic acid (IAA, 619pmolg -1 DW) and 2-oxindole-3-acetic acid (OxIAA, 2381pmolg -1 DW) were observed in EA-treated with NAA alone while mT treatment (without NAA) had the most abundant indole-3-acetyl-l-aspartic acid (IAAsp, 904 and 582pmolg -1 DW for EA and EZ, respectively) in both species. A significant concentration of total endogenous CK accumulated in both Eucomis regenerants from mT and mT+NAA when compared to the other treatments. The majority of the detected CKs were of the aromatic CK-type, mainly free bases. The potential physiological roles of these quantified phytohormones in relation to the observed morphological responses are discussed., (Copyright © 2016 Elsevier B.V. All rights reserved.)

TIR1 and its homologs act as auxin receptors and play important roles in plant growth and development in Arabidopsis thaliana. An auxin receptor homolog Solanum lycopersicum TIR1 (SlTIR1) has been isolated from tomato cultivar Micro-Tom, and SlTIR1 over-expression results in parthenocarpic fruit formation. In this study, the promoter driving the β-glucuronidase (GUS) expression vector was constructed and stably transformed into Micro-Tom seedlings. The SlTIR1 promoter driving GUS expression accumulated predominantly in the leaf and vasculature in transgenic seedlings. Promoter analysis identified an auxin-response element (AuxRE) and two gibberellic acid (GA)-response elements in the SlTIR1 promoter. Quantitative PCR showed that SlTIR1 transcript level was down-regulated by naphthaleneacetic acid, ethephon and abscisic acid and up-regulated by GA. Furthermore, because of the lack of ability to form reproductive seeds in SlTIR1 over-expressing Micro-Tom, this limits further exploration of potential roles of SlTIR1 in auxin signaling. Here, an antisense vector and an over-expression vector of the SlTIR1 gene were stably transformed into Micro-Tom and Ailsa Craig tomato, respectively. Phenotypes and physiological analyses indicated that SlTIR1 regulated primary root growth and auxin-associated lateral root formation in Micro-Tom. Meanwhile, SlTIR1 also stimulated abnormal seed development, so-called stenospermocarpy, in Ailsa Craig. Transcript accumulations of auxin-signaling genes determined by quantitative PCR were consistent with the idea that SlTIR1 regulated plant growth and development, partially mediated by controlling the mRNA levels of auxin-signaling genes. Our work demonstrates that SlTIR1 regulated auxin-induced root growth and stimulated stenospermocarpic fruit formation. SlTIR1 may be a key mediator of the crosstalk among auxin and other hormones to co-regulate plant growth and development., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Little is known about the transport mechanism of intracellular auxin. Here, we report two vacuole-localized proteins, Oryza sativa OsCONTINUOUS VASCULAR RING-LIKE 1 (OsCOLE1) and OsCOLE1-INTERACTING PROTEIN (OsCLIP), that regulate intracellular auxin transport and homoeostasis. Overexpression of OsCOLE1 markedly increased the internode length and auxin content of the stem base, whereas these parameters were decreased in RNA interference (RNAi) plants. OsCOLE1 was localized on the tonoplast and preferentially expressed in mature tissues. We further identified its interacting protein OsCLIP, which was co-localized on the tonoplast. Protein-protein binding assays demonstrated that the N-terminus of OsCOLE1 directly interacted with OsCLIP in yeast cells and the rice protoplast. Furthermore, (3) H-indole-3-acetic acid ((3) H-IAA) transport assays revealed that OsCLIP transported IAA into yeast cells, which was promoted by OsCOLE1. The results indicate that OsCOLE1 affects rice development by regulating intracellular auxin transport through interaction with OsCLIP, which provides a new insight into the regulatory mechanism of intracellular transport of auxin and the roles of vacuoles in plant development., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

Flurbiprofen axetil (FPA) is an injection product and a prodrug of a non-steroidal anti-inflammatory drug (NSAID). After injection, it is rapidly hydrolyzed to the active form, flurbiprofen (FP). Since frequent injections of FPA can lead to abnormal physiology, an administration strategy is necessary to ensure there is enhancement of the analgesic efficiency of FP after a single dose and to reduce the total number of doses. FP strongly binds to site II of albumin, and thus the free (unbound) FP concentration is low. This study focused on 6-methoxy-2-naphthylacetic acid (6-MNA), the active metabolite of nabumetone (a prodrug of NSAID). We performed ultrafiltration experiments and pharmacokinetics analysis in rats to investigate whether the inhibitory effect of 6-MNA on FP binding to albumin increased the free FP concentration in vitro and in vivo. Results indicated that 6-MNA inhibited the binding of FP to albumin competitively. When 6-MNA was injected in rats, there was a significant increase in the free FP concentration and the area under concentration-time curve (AUC) calculated from the free FP concentration, while there was a significant decrease in the total (bound + free) FP concentration and the AUC calculated from the total FP concentration. These findings indicate that 6-MNA inhibits the protein binding of FP in vivo. This suggests that the frequency of FPA injections can be reduced when administered with nabumetone, as there is increase in the free FP concentration associated with pharmacological effect.

Terminal shoot tips of sambong (Blumea balsamifera Linn.) are cultured to initiate and regenerate shoots on Murashige and Skoog (MS) medium containing 1.0 mg/L benzyl adenine (BA). After 1 month, shoots, usually 4.5 cm long are separated and subcultured for multiplication. Regenerated shoots, about 6 cm long are rooted on MS medium supplemented with 1.0 mg/L naphthalene acetic acid (NAA). Exposure of shoots to high humidity for the first 2 weeks and equal proportion (1:1:1) of sterile sand, compost, and coir dust as potting mix favors the development of whole sambong plants. Young shoots from in vitro-derived sambong plants could also be used for propagation.

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.

This paper concerns the studies towards membrane-damage effect of two auxins: indole-3-acetic acid - IAA and 1-naphthaleneacetic acid - NAA on plant (Arabidopsis thaliana) and animal (rat liver) model membranes. The foregoing auxins are plant growth regulators widely used in agriculture to control the quality of the crop. However, their accumulation in the environment makes them hazardous for the living organisms. The aim of our investigations was to compare the effect of natural (IAA) vs. synthetic (NAA) auxin on the organization of plant and animal model membranes and find a possible correlation between membrane-disturbing effect of these compounds and their toxicity. The collected data evidenced that auxins cause destabilization of membranes, decrease their condensation and weakens interactions of molecules. The alterations in the morphology of model systems were also noticed. The foregoing effects of auxins are concentration-dependent and additionally NAA was found to act on animal vs. plant membranes more selectively than IAA. Interestingly, both IAA and NAA induce the strongest disordering in model lipid system at the concentration, which is frequently reported as toxic to animal and plants. Based on the above findings it was proposed that membrane-damage effect induced by IAA and NAA may be important from the point of view of the mechanism of toxicity of these compounds and cannot be ignored in further investigations in this area., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Auxin-binding protein 1 (ABP1) is suggested to be an auxin receptor which plays an important role in several processes in green plants. Maize ABP1 was simulated with the natural auxin indole-3-acetic acid (IAA) and the synthetic analog naphthalen-1-acetic acid (NAA), to elucidate the role of the KDEL sequence and the helix at the C-terminus. The KDEL sequence weakens the intermolecular interactions between the monomers but stabilizes the C-terminal helix. Conformational changes at the C-terminus occur within the KDEL sequence and are influenced by the binding of the simulated ligands. This observation helps to explain experimental findings on ABP1 interactions with antibodies that are modulated by the presence of auxin, and supports the hypothesis that ABP1 acts as an auxin receptor. Stable hydrogen bonds between the monomers are formed between Glu40 and Glu62, Arg10 and Thr97, Lys39, and Glu62 in all simulations. The amino acids Ile22, Leu25, Trp44, Pro55, Ile130, and Phe149 are located in the binding pocket and are involved in hydrophobic interactions with the ring system of the ligand. Trp151 is stably involved in a face to end interaction with the ligand. The calculated free energy of binding using the linear interaction energy approach showed a higher binding affinity for NAA as compared to IAA. Our simulations confirm the asymmetric behavior of the two monomers, the stronger interaction of NAA than IAA and offers insight into the possible mechanism of ABP1 as an auxin receptor., (© 2014 Wiley Periodicals, Inc.)

Carbon-carbon bond cleavage reactions are catalyzed by, among others, lanosterol 14-demethylase (CYP51), cholesterol side-chain cleavage enzyme (CYP11), sterol 17β-lyase (CYP17), and aromatase (CYP19). Because of the high substrate specificities of these enzymes and the complex nature of their substrates, these reactions have been difficult to characterize. A CYP1A2-catalyzed carbon-carbon bond cleavage reaction is required for conversion of the prodrug nabumetone to its active form, 6-methoxy-2-naphthylacetic acid (6-MNA). Despite worldwide use of nabumetone as an anti-inflammatory agent, the mechanism of its carbon-carbon bond cleavage reaction remains obscure. With the help of authentic synthetic standards, we report here that the reaction involves 3-hydroxylation, carbon-carbon cleavage to the aldehyde, and oxidation of the aldehyde to the acid, all catalyzed by CYP1A2 or, less effectively, by other P450 enzymes. The data indicate that the carbon-carbon bond cleavage is mediated by the ferric peroxo anion rather than the ferryl species in the P450 catalytic cycle. CYP1A2 also catalyzes O-demethylation and alcohol to ketone transformations of nabumetone and its analogs.

Polar auxin transport is a crucial process for control and coordination of plant development. Studies of auxin transport through plant tissues and organs showed that auxin is transported by a combination of phloem flow and the active, carrier-mediated cell-to-cell transport. Since plant organs and even tissues are too complex for determination of the kinetics of carrier-mediated auxin uptake and efflux on the cellular level, simplified models of cell suspension cultures are often used, and several tobacco cell lines have been established for auxin transport assays. However, there are very few data available on the specificity and kinetics of auxin transport across the plasma membrane for Arabidopsis thaliana suspension-cultured cells. In this report, the characteristics of carrier-mediated uptake (influx) and efflux for the native auxin indole-3-acetic acid and synthetic auxins, naphthalene-1-acetic and 2,4-dichlorophenoxyacetic acids (NAA and 2,4-D, respectively) in A. thaliana ecotype Landsberg erecta suspension-cultured cells (LE line) are provided. By auxin competition assays and inhibitor treatments, we show that, similarly to tobacco cells, uptake carriers have high affinity towards 2,4-D and that NAA is a good tool for studies of auxin efflux in LE cells. In contrast to tobacco cells, metabolic profiling showed that only a small proportion of NAA is metabolized in LE cells. These results show that the LE cell line is a useful experimental system for measurements of kinetics of auxin carriers on the cellular level that is complementary to tobacco cells., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

In order to find a way to induce rooting on cuttings of Hemarthria compressa cv. Ya'an under controlled conditions, a project was carried out to study the effect of naphthalene acetic acid (NAA) on rooting in stem cuttings and related physiological changes during the rooting process of Hemarthria compressa. The cuttings were treated with five concentrations of NAA (0, 100, 200 300, 400 mg/l) at three soaking durations (10, 20, 30 minutes), and cuttings without treatment were considered as control. Samples were planted immediately into pots after treatment. IAA-oxidase (IAAO) activity, peroxidase (POD) activity and polyphenol oxidase (PPO) activity were determined after planting. Results showed that NAA had positive effect on rooting at the concentration of 200 mg/l compared to other concentrations at 30 days after planting (DAP). Among the three soaking durations, 20 minutes (min) of 200 mg/l NAA resulted in higher percentages of rooting, larger numbers of adventitious roots and heavier root dry weight per cutting. The lowest IAAO activity was obtained when soaked at 200 mg/l NAA for 20 min soaking duration. This was consistent with the best rooting ability, indicating that the lower IAAO activity, the higher POD activity and PPO activity could be used as an indicator of better rooting ability for whip grass cuttings and might serve as a good marker for rooting ability in cuttings.

Nabumetone is a non-acidic, nonsteroidal anti-inflammatory prodrug. Following oral administration, the prodrug is converted in the liver to 6-methoxy-2-naphthylacetic acid (6-MNA), which was found to be the principal metabolite responsible for the NSAID effect. The pathway of nabumetone transformation to 6-MNA has not been clarified, with no intermediates between nabumetone and 6-MNA having been identified to date. In this study, a new, as yet unreported phase I metabolite was discovered within the evaluation of nabumetone metabolism by human and rat liver microsomal fractions. Extracts from the biomatrices were subjected to chiral LLE-HPLC-PDA and achiral LLE-UHPLC-MS/MS analyses to elucidate the chemical structure of this metabolite. UHPLC-MS/MS experiments detected the presence of a structure corresponding to elemental composition C15H16O3, which was tentatively assigned as a hydroxylated nabumetone. Identical nabumetone and HO-nabumetone UV spectra obtained from the PDA detector ruled out the presence of the hydroxy group in the aromatic moiety of nabumetone. Hence, the most likely structure of the new metabolite was 4-(6-methoxy-2-naphthyl)-3-hydroxybutan-2-one (3-hydroxy nabumetone). To confirm this structure, the standard of this nabumetone metabolite was synthesized, its spectral (UV, CD, NMR, MS/MS) and retention properties on chiral and achiral chromatographic columns were evaluated and compared with those of the authentic nabumetone metabolite. To elucidate the subsequent biotransformation of 3-hydroxy nabumetone, the compound was used as a substrate in incubation with human and rat liver microsomal fraction. A number of 3-hydroxy nabumetone metabolites (products of conjugation with glucuronic acid, O-desmethylation, carbonyl reduction and their combination) were discovered in the extracts from the incubated microsomes using LLE-HPLC-PDA-MS/MS experiments. On the other hand, when 3-hydroxy nabumetone was incubated with isolated rat hepatocytes, 6-MNA was detected as the principal metabolite of 3-hydroxy nabumetone. Hence, 3-hydroxy nabumetone could be the missing link in nabumetone biotransformation to 6-MNA (i.e. nabumetone→3-hydroxy nabumetone→6-MNA)., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Auxin is one type of phytohormones that plays important roles in nearly all aspects of plant growth and developmental processes. The glycosylation of auxins is considered to be an essential mechanism to control the level of active auxins. Thus, the identification of auxin glycosyltransferases is of great significance for further understanding the auxin regulation. In this study, we biochemically screened the group L of Arabidopsis thaliana glycosyltransferase superfamily for enzymatic activity toward auxins. UGT74D1 was identified to be a novel auxin glycosyltransferase. Through HPLC and LC-MS analysis of reaction products in vitro by testing eight substrates including auxins and other compounds, we found that UGT74D1 had a strong glucosylating activity toward indole-3-butyric acid [IBA], indole-3-propionic acid [IPA], indole-3-acetic acid [IAA] and naphthaleneacetic acid [NAA], catalyzing them to form corresponding glucose esters. Biochemical characterization showed that this enzyme had a maximum activity in HEPES buffer at pH 6.0 and 37°C. In addition, the enzymatic activity analysis of crude protein and the IBA metabolite analysis from transgenic Arabidopsis plants overexpressing UGT74D1 gene were also carried out. Experimental results indicated that over-production of the UGT74D1 in plants indeed led to increased level of the glucose conjugate of IBA. Moreover, UGT74D1 overexpression lines displayed curling leaf phenotype, suggesting a physiological role of UGT74D1 in affecting the activity of auxins. Our current data provide a new target gene for further genetic studies to understand the auxin regulation by glycosylation in plants.

KEY MESSAGE : Rooting of Artemisia annua increases trichome size on leaves and helps drive the final steps of the biosynthesis of the sesquiterpene antimalarial drug, artemisinin. Artemisia annua produces the antimalarial drug, artemisinin (AN), which is synthesized and stored in glandular trichomes (GLTs). In vitro-grown A. annua shoots produce more AN when they form roots. This may be a function not of the roots, but rather media components such as the phytohormones, α-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP), or salts and sucrose used to maintain either rooted or unrooted shoot cultures. We investigated how three main media components altered artemisinic metabolite production, pathway gene transcripts, and GLT formation in both mature and developing leaves in rooted and unrooted cultures. Although transcript levels of AN biosynthetic genes were not altered, AN levels were significantly different, and there were major differences in both artemisinic metabolite levels and trichomes in mature versus developing leaves. For example, NAA induced higher AN production in rooted shoots, but only in mature leaves. In developing leaves, BAP increased GLT density on the leaf surface. When both phytohormones were present, GLTs were larger on young developing leaves, but smaller on mature leaves. Furthermore, although other media components increased GLT density, their size decreased on young leaves, but there was no effect on mature leaves. Roots also appeared to drive conversion of artemisinic precursors towards end products. These results suggest that, while the presence of roots affects AN and trichome production, phytohormones and other media constituents used for in vitro culture of A. annua also exert an influence.

Chemical pesticides have been widely used to increase the yield and quality of agricultural products as they are efficient, effective, and easy to apply. However, the rapid degradation and low utilization ratio of conventional pesticides has led to environmental pollution and resource waste. Nano-sized controlled-release formulations (CRFs) can provide better penetration through the plant cuticle and deliver the active ingredients efficiently to the targeted tissue. In this paper we reported novel conjugated nanospheres derived from 1-naphthylacetic acid (NNA), 3-aminopropyltriethoxysilane (APTES) and tetraethyl orthosilicate and their application as a controlled-release plant growth regulator. The NNA and APTES conjugate was prepared through a covalent cross-linking reaction and subsequent hydrolyzation and polycondensation to synthesize NNA-silica nanospheres. The release data indicated that the release of NNA was by non-Fickian transport and increased as particle size decreased. It was also found that the acidity-alkalinity was enhanced and as the temperature increased, the release of the active ingredient was faster. The nanoconjugate displayed a better efficacy in promoting root formation than NNA technical. The present study provides a novel synthesis route for CRFs comprising a pesticide, with long-duration sustained-release performance and good environmental compatibility. This method may be extended to other pesticides that possess a carboxyl group.

Flower opening in Iris (Iris×hollandica) requires elongation of the pedicel and ovary. This moves the floral bud upwards, thereby allowing the tepals to move laterally. Flower opening is requires with elongation of the pedicel and ovary. In cv. Blue Magic, we investigated the possible role of hormones other than ethylene in pedicel and ovary elongation and flower opening. Exogenous salicylic acid (SA) and the cytokinins benzyladenine (N6-benzyladenine, BA) and zeatin did not affect opening. Jasmonic acid (JA) and abscisic acid (ABA) were slightly inhibitory, but an inhibitor of ABA synthesis (norflurazon) was without effect. Flower opening was promoted by gibberellic acid (GA(3)), but two inhibitors of gibberellin synthesis (4-hydroxy-5-isopropyl-2-methylphenyltrimethyl ammonium chloride-1-piperidine carboxylate, AMO-1618; ancymidol) did not change opening. The auxins indoleacetic acid (IAA) and naphthaleneacetic acid (NAA) strongly promoted elongation and opening. An inhibitor of auxin transport (2,3,5-triodobenzoic acid, TIBA) and an inhibitor of auxin effects [α-(p-chlorophenoxy)-isobutyric acid; PCIB] inhibited elongation and opening. The data suggest that endogenous auxins are among the regulators of the pedicel and ovary elongation and thus of flower opening in Iris., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

The phytohormone auxin is critical for plant growth and many developmental processes. Members of the P-glycoprotein (PGP/ABCB) subfamily of ATP-binding cassette (ABC) transporters have been shown to function in the polar movement of auxin by transporting auxin over the plasma membrane in both monocots and dicots. Here, we characterize a new Arabidopsis member of the ABCB subfamily, ABCB21/PGP21, a close homolog of ABCB4, for which conflicting transport directionalities have been reported. ABCB21 is strongly expressed in the abaxial side of cotyledons and in junctions of lateral organs in the aerial part, whereas in roots it is specifically expressed in pericycle cells. Membrane fractionation by sucrose density gradient centrifugation followed by Western blot showed that ABCB21 is a plasma membrane-localized ABC transporter. A transport assay with Arabidopsis protoplasts suggested that ABCB21 was involved in IAA transport in an outward direction, while naphthalene acetic acid (NAA) was a less preferable substrate for ABCB21. Further functional analysis of ABCB21 using yeast import and export assays showed that ABCB21 mediates the 1-N-naphthylphthalamic acid (NPA)-sensitive translocation of auxin in an inward direction when the cytoplasmic IAA concentration is low, whereas this transporter mediates outward transport under high internal IAA. An increase in the cytoplasmic IAA concentration by pre-loading of IAA into yeast cells abolished the IAA uptake activity by ABCB21 as well as ABCB4. These findings suggest that ABCB21 functions as a facultative importer/exporter controlling auxin concentrations in plant cells.

Objective: To screen out fungus strains with acetylcholinesterase inhibitory activity from Huperzia serrata., Method: Endophytic fungi fermentation products from 59 H. serrata strains were stained with acetylcholinesterase hydrolyzed alpha-naphthaleneacetic ethyl ester and fast blue B salt, and screened for acetylcholinesterase inhibitory activity with thin-layer chromatography-bioautography. Target strains were classified and identified through the sequence analysis on 18s rDNA and 5.8s rDNA combined with morphological characteristics., Result: Fungus strain LQ2F01 from H. serrata showed positive color reaction in the screening for acetylcholinesterase inhibitory activity. The sequence analysis on 18s rDNA and 5.8s rDNA combined with morphological characteristics showed the strain LQ2F01 belonged to Acremonium., Conclusion: Endophytic Fungi LQ2F01 from H. serrata shows identical acetylcholinesterase inhibitory activity with the host plant, which is of great significance to the development of natural medicines and the studies on the relationship between the endophytic gungi and the host plant.

A simple fluorescent methodology for the simultaneous determination of nabumetone and its main metabolite, 6-methoxy-2-naphthylacetic acid (6-MNA), in pharmaceutical preparations and human urine is proposed. Due to the strong overlapping between the fluorescence spectra of both analytes, the use of fluorescence decay curves to resolve their mixture is proposed, since these curves are more selective. Values of dependent instrumental variables affecting the signal-to-noise ratio were fixed using a simplex optimization procedure. A factorial design with three levels per factor coupled to a central composite design was selected to obtain a calibration matrix of thirteen standards plus one blank sample that was processed using a partial least-squares (PLS) analysis. In order to assess the goodness of the proposed method, a prediction set of ten synthetic samples was analyzed, obtaining recovery percentages between 97 and 105%. Limits of detection, calculated by means of a new criterion, were 0.96 μg L(-1) and 0.88 μg L(-1) for nabumetone and 6-MNA, respectively. The method was also tested in the pharmaceutical preparation Relif, which contains nabumetone, obtaining recovery percentages close to 100%. Finally, the simultaneous determination of both analytes in human urine samples was successfully carried out by the PLS-analysis of a matrix of fifteen standards plus four analyte blanks and the use of the standard addition technique. Although urine shows native fluorescence, no extraction method or prior separation of the analytes was needed.

The effect on endoreduplication in sugar beet (Beta vulgaris L.) seedlings of five plant hormones in MS medium, ethylene, 24-epibrassinolide (EBR), gibberellic acid (GA(3) ), kinetin and 1-naphthaleneacetic acid (NAA), as well as a combination of kinetin and NAA at two different concentrations, was studied using flow cytometry. Analyses of DNA content in nuclei of the root, hypocotyl and cotyledons of seedlings growing in vitro were performed during their early development, starting from when the root was 0.5-1.0 cm long until expansion of the first pair of leaves. The proportions of nuclei with different DNA contents were established and the mean C-value calculated. The presence of exogenous plant hormones changed endoreduplication intensity, although to different extents, depending on the organ and developmental stage. Ethylene and NAA stimulated the process, while EBR and kinetin suppressed it and GA did not clearly affect it., (© 2011 German Botanical Society and The Royal Botanical Society of the Netherlands.)

Ester precursors of fluorogenic or chromogenic probes are often employed in studies of yeast cell biology. This study was aimed at a comparison of the ability of several commonly used laboratory wild-type Saccharomyces cerevisiae strains to hydrolyse the following model esters: fluorescein diacetate, 2-naphthyl acetate, PNPA (p-nitrophenyl acetate) and AMQI (7-acetoxy-1-methylquinolinum iodide). In all the strains, the esterase activity was localized mainly to the cytosol. Considerable differences in esterase activity were observed between various wild-type laboratory yeast strains. The phase of growth also contributed to the variation in esterase activity of the yeast. This diversity implies the need for caution in using intracellularly hydrolysed probes for a comparison of yeast strains with various genetic backgrounds.

Background: Naphthaleneacetic acid (NAA), a synthetic auxin analogue, is widely used as an effective thinner in apple orchards. When applied shortly after fruit set, some fruit abscise leading to improved fruit size and quality. However, the thinning results of NAA are inconsistent and difficult to predict, sometimes leading to excess fruit drop or insufficient thinning which are costly to growers. This unpredictability reflects our incomplete understanding of the mode of action of NAA in promoting fruit abscission., Results: Here we compared NAA-induced fruit drop with that caused by shading via gene expression profiling performed on the fruit abscission zone (FAZ), sampled 1, 3, and 5 d after treatment. More than 700 genes with significant changes in transcript abundance were identified from NAA-treated FAZ. Combining results from both treatments, we found that genes associated with photosynthesis, cell cycle and membrane/cellular trafficking were downregulated. On the other hand, there was up-regulation of genes related to ABA, ethylene biosynthesis and signaling, cell wall degradation and programmed cell death. While the differentially expressed gene sets for NAA and shading treatments shared only 25% identity, NAA and shading showed substantial similarity with respect to the classes of genes identified. Specifically, photosynthesis, carbon utilization, ABA and ethylene pathways were affected in both NAA- and shading-induced young fruit abscission. Moreover, we found that NAA, similar to shading, directly interfered with leaf photosynthesis by repressing photosystem II (PSII) efficiency within 10 minutes of treatment, suggesting that NAA and shading induced some of the same early responses due to reduced photosynthesis, which concurred with changes in hormone signaling pathways and triggered fruit abscission., Conclusions: This study provides an extensive transcriptome study and a good platform for further investigation of possible regulatory genes involved in the induction of young fruit abscission in apple, which will enable us to better understand the mechanism of fruit thinning and facilitate the selection of potential chemicals for the thinning programs in apple.

In response to Fe-deficiency, various dicots increase their root branching which contributes to the enhancement of ferric-chelate reductase activity. Whether this Fe-deficiency-induced response eventually enhances the ability of the plant to tolerate Fe-deficiency or not is still unclear and evidence is also scarce about the signals triggering it. In this study, it was found that the SPAD-chlorophyll meter values of newly developed leaves of four tomato (Solanum lycocarpum) lines, namely line227/1 and Roza and their two reciprocal F(1) hybrid lines, were positively correlated with their root branching under Fe-deficient conditions. It indicates that Fe-deficiency-induced root branching is critical for plant tolerance to Fe-deficiency. In another tomato line, Micro-Tom, the increased root branching in Fe-deficient plants was accompanied by the elevation of endogenous auxin and nitric oxide (NO) levels, and was suppressed either by the auxin transport inhibitors NPA and TIBA or the NO scavenger cPTIO. On the other hand, root branching in Fe-sufficient plants was induced either by the auxin analogues NAA and 2,4-D or the NO donors NONOate or SNP. Further, in Fe-deficient plants, NONOate restored the NPA-terminated root branching, but NAA did not affect the cPTIO-terminated root branching. Fe-deficiency-induced root branching was inhibited by the NO-synthase (NOS) inhibitor L-NAME, but was not affected by the nitrate reductase (NR) inhibitor NH(4)(+), tungstate or glycine. Taking all of these findings together, a novel function and signalling pathway of Fe-deficiency-induced root branching is presented where NOS-generated rather than NR-generated NO acts downstream of auxin in regulating this Fe-deficiency-induced response, which enhances the plant tolerance to Fe-deficiency.

Objective: To study the effects of different auxcin regulators on rooting of Periplocaforrestii cuttings as well as dynamic change rules of endogenous plant hormones and oxidases related to adventitious root formation., Methods: Cuttings propagation characters of Periploca forrestii were investigated and compared with different concentration treatments of indolebutyric acid (IBA), Rooting Powder No. 1 (ABT1) and naphthylacetic acid (NAA). The dynamic changes of contents of endogenous hormones including indole acetic acid (IAA), abscisic acid (ABA), zeatin ribosides (ZRs) as well as the activities of indoleacetic acid oxidase( IAAO), polyphenol oxidase (PPO), peroxidase (POD) were tested., Results: Rooting percentage of cutting with 150 mg/L IBA, 150 mg/L ABT1 treatment and NAA treatment were 80% ,70% and 68% respectively, rooting percentage of cuttings of the control was 23% only. The adventitious rooting displayed three distinct phases i. e. root-inducing, root-formating and root-elongating phases. During root-inducing phase the contents of IAA, ABA and ZRs decreased,whereas IAAO activity kept at a higher level. The IAA content reached the peak and PPO activity increased obviously during root-formating phase, while activities of IAAO, POD and contents of ABA, ZRs declined to minimum. During root-elongating phase contents of IAA, ABA, ZRs were much steadfast and activities of PPO, IAAO, POD were increased. After that, the activities of the three oxidases decreased slowly. 150 mg/L IBA treatment increased the content of IAA and PPO activity in cuttings during rooting,while the opposite result occurred in contents of ZRs, ABA and the activities of IAAO, POD., Conclusion: The dynamic changes of endogenous hormones (IAA, ABA, ZRs) contents and IAAO, PPO, POD activities are tightly related to the rooting process of cuttings in Periploca forrestii.

The cytochrome P450 (CYP) isoforms that catalyze the oxidation metabolism of 6-methoxy-2-napthylacetic acid (6-MNA), an active metabolite of nabumetone, were studied in rats and humans. Using an extractive reversed-phase HPLC assay with fluorescence detection, monophasic Michaelis-Menten kinetics was obtained for the formation of 6-hydroxy-2-naphthylacetic acid (6-HNA) in liver microsomes of rats and humans, and kinetic analysis showed that the K(m) and V(max) values for the formation of 6-HNA in humans and rats were 640.0 ± 30.9 and 722.9 ± 111.7 µM, and 1167.5 ± 33.0 and 1312.7 ± 73.8 pmol min⁻¹ mg protein⁻¹, respectively. The CYPs responsible for metabolism of 6-MNA in liver microsomes of rats and humans were identified using correlation study, recombinant CYP supersomes, and specific CYP inhibitors and antibodies. Recombinant human CYP2C9 exhibited appreciable catalytic activity with respect to 6-HNA formation from 6-MNA. Among 14 recombinant rat CYPs examined, CYP2C6, CYP2C11 and CYP1A2 were involved in the metabolism of 6-MNA. Sulfaphenazole (a selective inhibitor of CYP2C9) inhibited the formation of 6-HNA in pooled human microsomes by 89%, but failed to inhibit this reaction in rat liver microsomes. The treatment of pooled human liver microsomes with an antibody against CYP2C9 inhibited the formation of 6-HNA by about 80%. The antibody against CYP2C11 suppressed the activity by 20 to 30% in rat microsomes, whereas that of CYP1A2 microsomes did not show drastic inhibition. These findings suggest that CYP2C9 has the highest catalytic activity of 6-MNA metabolism in humans. In contrast, metabolism of 6-MNA is suggested to be mediated mainly by CYP2C6 and CYP2C11 in rats.

The synthesis of cell wall polysaccharides is highly active in rapidly growing bamboo shoots. We cloned a set of BoCesA cDNAs that encode cellulose synthase from bamboo (Bambusa oldhamii) and investigated the expression patterns of the BoCesA2, BoCesA5, BoCesA6 and BoCesA7 genes. The four BoCesA genes were differentially expressed in the different parts of growing bamboo shoots, in various organs, and in multiple shoots that were cultured in vitro. They were down-regulated by alpha-naphthaleneacetic acid and differentially affected by thidiazuron in the multiple shoots. In situ RT-PCR analyses demonstrated that BoCesA2, BoCesA5, BoCesA6, and BoCesA7 mRNAs were present throughout the base and the internode regions of the etiolated shoots that emerged from pseudorhizomes, and in the internode regions of the juvenile branch shoots that emerged from nodes of mature bamboo culms; however, the expression of the four genes in the lignified internode of the branch shoot was predominantly detected in the center of the vascular bundles. Our results for cDNA cloning, expression analyses, and phylogenetic analysis suggest that the 10 BoCesA genes cloned from the etiolated bamboo shoots participate in cellulose synthesis in the primary cell walls of the growing bamboo, and that at least three additional BoCesA genes involved in cellulose synthesis in the secondary walls may be present in the bamboo genome. The expressions of BoCesA genes may be under fine control in response to the various developmental stages and physiological conditions of bamboo., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Objective: To study the effect of the different constitutions of plant hormone on the development of Anoectochilus roxburghii., Method: A. roxburghii were harvested after having been cultured for 60 days. An orthogonal design was used to study the effect of NAA and 6-BA on the leaf number, eustipe number, lateral branch number of the stem tip and stem section, and the height of the stem tips. All of the data were processed by SPSS., Result and Conclusion: It is reported for the first time that NAA could make different development of A. roxburghii at low concentration ( < 1 mg L(-1)) and high concentration ( > 1 mg L(-1)). The optimum constitution of MS medium was NAA 0.5 mg L(-1) + 6-BA 1 mg L(-1) for the growth of the stem tip of A. roxburghii, and NAA 1 mg L(-1) + 6-BA 2 mg L(-1) for the differentiation of bud and the formation of lateral branch of the stem section. The different concentrations of NAA and 6-BA had different effects on the growth and differentiation of the stem tip and the stem section of A. roxburghii.

Use of a synthetic auxin (naphthalene-1-acetic acid, NAA) to start (Gossypium hirsutum) ovule/fiber cultures hindered fiber secondary wall cellulose synthesis compared with natural auxin (indole-3-acetic acid, IAA). In contrast, NAA promoted fiber elongation and ovule weight gain, which resulted in larger ovule/fiber units. To reach these conclusions, fiber and ovule growth parameters were measured and cell wall characteristics were examined microscopically. The differences in fiber from NAA and IAA culture were underpinned by changes in the expression patterns of marker genes for three fiber developmental stages (elongation, the transition stage, and secondary wall deposition), and these gene expression patterns were also analyzed quantitatively in plant-grown fiber. The results demonstrate that secondary wall cellulose synthesis: (1) is under strong transcriptional control that is influenced by auxin; and (2) must be specifically characterized in the cotton ovule/fiber culture system given the many protocol variables employed in different laboratories.

Introduction: The binding of radiopharmaceutical to serum proteins is thought to be an important factor that restricts its excretion and accumulation in tissue. We calculated the effect of inhibitors of serum protein binding using a hypothetical radiopharmaceutical. In vitro experiments and protein binding inhibitor-loaded monkey scintigraphy were then conducted using (123)I-N-isopropyl-p-iodoamphetamine (IMP) as the radiopharmaceutical., Methods: Free fraction ratios of radiopharmaceutical were calculated with one radiopharmaceutical, two serum proteins and two specific inhibitors in the steady state at various serum protein concentrations. In vitro protein binding inhibition studies using human, rat and monkey sera were performed with site-selective displacers of specific binding sites: 400 microM 6-methoxy-2-naphthylacetic acid (6MNA; a major nabumeton metabolite) as a serum albumin Site II inhibitor and 400 microM erythromycin (ETC) as an alpha(1)-acid glycoprotein (AGP) site inhibitor. Scintigraphy with or without 6MNA loading of monkeys was performed., Results: The theoretical findings roughly corresponded to the experimental results. Approximately 75% of IMP bound to serum albumin Site II and AGP in the species examined. The free fraction of IMP (25.0+/-0.6% for human, 22.8+/-0.4% for monkey, 23.7+/-0.3% for rat) increased with loading of specific protein binding inhibitors (6MNA: 28.0+/-0.3% for human, 24.5+/-0.7% for monkey, 24.3+/-0.2% for rat; ETC: 26.3+/-0.4% for human, 29.5+/-1.1% for monkey, 26.0+/-0.7% for rat) and was serum protein concentration dependant based on the results of calculations. Simultaneous administration of 6MNA and ETC produced a higher free fraction ratio of IMP (31.9+/-1.0% for human, 34.6+/-0.4% for monkey, 27.0+/-0.3% for rat) than summation of the single administrations of 6MNA and ETC (domino effect) in human, rat and monkey sera. Rapid cerebral accumulation was observed with 6MNA loading in monkey scintigraphy., Conclusions: 6MNA appears to change the pharmacokinetics and brain accumulation of IMP in monkeys. Further studies in human are required.

A simple, precise and accurate assay for the determination of 6-methoxy-2-naphthylacetic acid (6-MNA), an active metabolite of nabumetone in human plasma, was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The analyte (6-MNA) and propranolol (internal standard, IS) were extracted from 200 microL aliquot of human plasma via solid-phase extraction employing HLB Oasis cartridges and separated on a Discovery HS C18 (50 x 4.6 mm, 5 microm) column. Detection of analyte and IS was done by tandem mass spectrometry with a turbo ion spray interface operating in positive ion and multiple reaction monitoring acquisition mode. The total chromatographic runtime was 3.0 min with retention time for 6-MNA and IS at 1.97 and 1.26 min, respectively. The method was validated over a dynamic linear range of 0.20-60.00 microg/mL for 6-MNA with mean correlation coefficient r > or = 0.9986. The intra-batch and inter-batch precision (%CV) across five validation runs (lower limit of quantiation, low-, medium- and high-quality controls and upper limit of quantitation) was less than 7.5%. The accuracy determined at these levels was within -5.8 to +0.2% in terms of percentage bias. The method was successfully applied for a bioequivalence study of 750 mg nabumetone tablet formulation in 12 healthy Indian male subjects under fasted condition.

Nitrate reductase (NR), a committed enzyme in nitrate assimilation, involves generation of nitric oxide (NO) in plants. Here we show that the NR activity was significantly enhanced by the addition of NO donors sodium nitroprusside (SNP) and NONOate (diethylamine NONOate sodium) to the culturing solution, whereas it was decreased by NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (cPTIO). Interestingly, both NO gas and SNP directly enhanced but cPTIO inhibited the NR activities of crude enzyme extracts and purified NR enzyme. The cPTIO terminated the interaction between NR-generated NO and the NR itself. Furthermore, the NR protein content was not affected by the SNP treatment. The investigation of the partial reactions catalysed by purified NR using various electron donors and acceptors indicated that the haem and molybdenum centres in NR were the two sites activated by NO. The results suggest that the activation of NR activity by NO is regulated at the post-translational level, probably via a direct interaction mechanism. Accordingly, the concentration of nitrate both in leaves and roots was decreased after 2 weeks of cultivation with SNP. The present study identifies a new mechanism of NR regulation and nitrate assimilation, which provides important new insights into the complex regulation of N-metabolism in plants.

We report the first metabolic labeling of Arabidopsis thaliana for proteomic investigation, demonstrating efficient and complete labeling of intact plants. Using a reversed-database strategy, we evaluate the performance of the MASCOT search engine in the analysis of combined natural abundance and 15N-labeled samples. We find that 15N-metabolic labeling appears to increase the ambiguity associated with peptide identifications due in part to changes in the number of isobaric amino acids when the isotopic label is introduced. This is reflected by changes in the distributions of false positive identifications with respect to MASCOT score. However, by determining the nitrogen count from each pair of labeled and unlabeled peptides we may improve our confidence in both heavy and light identifications.

Intercellular flow of the phytohormone auxin underpins multiple developmental processes in plants. Plant-specific pin-formed (PIN) proteins and several phosphoglycoprotein (PGP) transporters are crucial factors in auxin transport-related development, yet the molecular function of PINs remains unknown. Here, we show that PINs mediate auxin efflux from mammalian and yeast cells without needing additional plant-specific factors. Conditional gain-of-function alleles and quantitative measurements of auxin accumulation in Arabidopsis and tobacco cultured cells revealed that the action of PINs in auxin efflux is distinct from PGP, rate-limiting, specific to auxins, and sensitive to auxin transport inhibitors. This suggests a direct involvement of PINs in catalyzing cellular auxin efflux.