Lupeol Accumulation Correlates with Auxin in the Epidermis of Castor.

Lupeol, a natural lupane-type pentacyclic triterpene, possesses various pharmacological properties, and its production attracts attention. Significant quantities of lupeol are deposited on the castor aerial organ surface and are easily extractable as a predominant wax constituent. Thus, castor might be considered as a potential bioreactor for the production of lupeol. The lupeol biosynthesis pathway is well known, but how it is regulated remains largely unknown. Among large numbers of castor cultivars, we targeted one accession line (337) with high levels of lupeol on its stem surface and low levels thereof on its hypocotyl surface, implicating that lupeol synthesis is differentially regulated in the two organs. To explore the underlying mechanisms, we did comparative transcriptome analysis of the first internode of 337 stem and the upper hypocotyl. Our results show that large amounts of auxin-related genes are differentially expressed in both parts, implying some possible interactions between auxin and lupeol production. We also found that several auxin-responsive cis-elements are present in promoter regions of HMGR and LUS genes encoding two key enzymes involved in lupeol production. Furthermore, auxin treatments apparently induced the expression levels of RcHMGR and RcLUS. Furthermore, we observed that auxin treatment significantly increased lupeol contents, whereas inhibiting auxin transport led to an opposite phenotype. Our study reveals some relationships between hormone activity and lupeol synthesis and might provide a promising way for improving lupeol yields in castor.

Reinvestigation of THOUSAND-GRAIN WEIGHT 6 grain weight genes in wheat and rice indicates a role in pollen development rather than regulation of auxin content in grains.

KEY MESSAGE: Phylogenetic and expression analyses of grain weight genes TaTGW6 and OsTGW6 and investigation of substrate availability indicate TGW6 does not regulate auxin content of grains but may affect pollen development. The THOUSAND-GRAIN WEIGHT 6 genes (TaTGW6 and OsTGW6) are reported to result in larger grains of wheat and rice by reducing production of indole-3-acetic acid (IAA) in developing grains. However, a critical comparison of data on TaTGW6 and OsTGW6 with other reports on IAA synthesis in cereal grains requires that this hypothesis be reinvestigated. Here, we show that TaTGW6 and OsTGW6 are members of a large gene family that has undergone major, lineage-specific gene expansion. Wheat has nine genes, and rice three genes encoding proteins with more than 80% amino acid identity with TGW6, making it difficult to envisage how a single inactive allele could have a major effect on IAA levels in grains. In our study, we show that neither TaTGW6 nor OsTGW6 is expressed in developing grains. Instead, both genes and their close homologues are exclusively expressed in pre-emergent inflorescences; TaTGW6 is expressed particularly in microspores prior to mitosis. This evidence, combined with our observation that developing wheat grains have undetectable levels of ester IAA in comparison to free IAA and do not express an IAA-glucose synthase suggests that TaTGW6 and OsTGW6 do not regulate grain size via the hydrolysis of IAA-glucose. Instead, their similarity to rice strictosidine synthase-like (OsSTRL2) suggests they play a key role in pollen development.

Volatile 1-octanol of tea (Camellia sinensis L.) fuels cell division and indole-3-acetic acid production in phylloplane isolate Pseudomonas sp. NEEL19.

Tea leaves possess numerous volatile organic compounds (VOC) that contribute to tea's characteristic aroma. Some components of tea VOC were known to exhibit antimicrobial activity; however, their impact on bacteria remains elusive. Here, we showed that the VOC of fresh aqueous tea leaf extract, recovered through hydrodistillation, promoted cell division and tryptophan-dependent indole-3-acetic acid (IAA) production in Pseudomonas sp. NEEL19, a solvent-tolerant isolate of the tea phylloplane. 1-octanol was identified as one of the responsible volatiles stimulating cell division, metabolic change, swimming motility, putative pili/nanowire formation and IAA production, through gas chromatography-mass spectrometry, microscopy and partition petri dish culture analyses. The bacterial metabolic responses including IAA production increased under 1-octanol vapor in a dose-dependent manner, whereas direct-contact in liquid culture failed to elicit such response. Thus, volatile 1-octanol emitting from tea leaves is a potential modulator of cell division, colonization and phytohormone production in NEEL19, possibly influencing the tea aroma.

Endogenous levels of cytokinins, indole-3-acetic acid and abscisic acid in in vitro grown potato: A contribution to potato hormonomics.

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

Analytical Determination of Auxins and Cytokinins.

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

Indole acetic acid and ACC deaminase from endophytic bacteria improves the growth of Solarium lycopersicum

Background: Endophytic bacteria are ubiquitous in all plant species contributing in host plant's nutrient uptake and helping the host to improve its growth. Moringa peregrina which is a medicinal plant, growing in arid region of Arabia, was assessed for the presence of endophytic bacterial strains. Results: PCR amplification and sequencing of 16S rRNA of bacterial endophytes revealed the 5 endophytic bacteria, in which 2 strains were from Sphingomonas sp.; 2 strains from Bacillus sp. and 1 from Methylobacterium genus. Among the endophytic bacterial strains, a strain of Bacillus subtilis LK14 has shown significant prospects in phosphate solubilization (clearing zone of 56.71 mm after 5 d), ACC deaminase (448.3 ± 2.91 nM α-ketobutyrate mg-1 h-1) and acid phosphatase activity (8.4 ± 1.2 nM mg-1 min-1). The endophytic bacteria were also assessed for their potential to produce indole-3-acetic acid (IAA). Among isolated strains, the initial spectrophotometry analysis showed significantly higher IAA production by Bacillus subtilis LK14. The diurnal production of IAA was quantified using multiple reactions monitoring method in UPLC/MS-MS. The analysis showed that LK14 produced the highest (8.7 uM) IAA on 14th d of growth. Looking at LK14 potentials, it was applied to Solanum lycopersicum, where it significantly increased the shoot and root biomass and chlorophyll (a and b) contents as compared to control plants. Conclusion: The study concludes that using endophytic bacterial strains can be bio-prospective for plant growth promotion, which might be an ideal strategy for improving growth of crops in marginal lands.

Effects of interactions of auxin-producing bacteria and bacterial-feeding nematodes on regulation of peanut growths.

The influences of an IAA (indole-3-acetic acid)-producing bacterium (Bacillus megaterium) and two bacterial-feeding nematodes (Cephalobus sp. or Mesorhabditis sp.) on the growth of peanut (Arachis hypogaea L. cv. Haihua 1) after various durations of time were investigated in natural soils. The addition of bacteria and nematodes and incubation time all significantly affected plant growth, plant root growth, plant nutrient concentrations, soil nutrient concentrations, soil microorganisms and soil auxin concentration. The addition of nematodes caused greater increases in these indices than those of bacteria, while the addition of the combination of bacteria and nematodes caused further increases. After 42-day growth, the increases in soil respiration differed between the additions of two kinds of nematodes because of differences in their life strategies. The effects of the bacteria and nematodes on the nutrient and hormone concentrations were responsible for the increases in plant growth. These results indicate the potential for promoting plant growth via the addition of nematodes and bacteria to soil.

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.

Development of a rapid LC-DAD/FLD method for the simultaneous determination of auxins and abscisic acid in plant extracts.

Plant hormones play a crucial role in controlling plant growth and development. These groups of naturally occurring substances trigger physiological processes at very low concentrations, which mandate sensitive techniques for their quantitation. This paper describes a method to quantify endogenous (±)-2-cis-4-trans-abscisic acid, indole-3-acetic acid, indole-3-propionic acid, and indole-3-butyric acid. The method combines high-performance liquid chromatography (HPLC) with diode array and fluorescence detection in a single run. Hybrid tea rose 'Monferrato' matrices (leaves, petals, roots, seeds, androecium, gynoecium, and pollen) were used as references. Rose samples were separated and suspended in extracting methanol, after which (±)-2-cis-4-trans-abscisic acid and auxins were extracted by solvent extraction. Sample solutions were added first to cation solid phase extraction (SPE) cartridges and the eluates to anion SPE cartridges. The acidic hormones were bound to the last column and eluted with 5% phosphoric acid in methanol. Experimental results showed that this approach can be successfully applied to real samples and that sample preparation and total time for routine analysis can be greatly reduced.

Quantification of abscisic Acid, cytokinin, and auxin content in salt-stressed plant tissues.

Plant hormones cytokinins, auxin (indole-3-acetic acid), and abscisic acid are central to regulation of plant growth and defence to abiotic stresses such as salinity. Quantification of the hormone levels and determination of their ratios can reveal different plant strategies to cope with the stress, e.g., suppression of growth or mobilization of plant metabolism. This chapter describes a procedure enabling such quantification. Due to the high variability of these hormones in plant tissues, it is advantageous to determine their content in the same sample. Reverse phase and ion exchange chromatography allows separation of the individual hormone fractions. Hormones as well as their metabolites can be identified and quantified by LC/MS.

Rapid and sensitive detection of auxins and flavonoids in plant samples by high-performance liquid chromatography coupled with tandem mass spectrometry.

Simultaneous determination of indole-3-acetic acid and methyl indole-3-acetic acid ester in small amounts of plant tissue is essential for elucidating their mutual transformation mechanism and the in vivo function of methyl indole-3-acetic acid ester. Rapid quantification of flavonoids in the same sample is important for clarifying their roles in the transport of auxins and other phytohormones. Herein, we describe a simple method for the simultaneous determination of indole-3-acetic acid and its methyl ester in the roots of the Arabidopsis thaliana seedlings and a protocol for the rapid extraction and quantification of quercetin and kaempferol in these seedlings. High-performance liquid chromatography coupled with electrospray ionization time-of-flight tandem mass spectrometry was used for the detection of all the compounds. Negative data for indole-3-acetic acid and positive data for methyl indole-3-acetic acid ester were collected in two successive files with a single injection of the extracted sample. Under optimized conditions, the limit of detection for the four compounds was 2 ng/mL for indole-3-acetic acid, 0.5 ng/mL for methyl indole-3-acetic acid ester, 5 ng/mL for quercetin, and 1 ng/mL for kaempferol, respectively. Because of the high sensitivity of the assay, only 2-10 mg of the plant material was required to obtain quantitative results.

The effects of auxin and strigolactones on tuber initiation and stolon architecture in potato.

Various transcriptional networks and plant hormones have been implicated in controlling different aspects of potato tuber formation. Due to its broad impact on many plant developmental processes, a role for auxin in tuber initiation has been suggested but never fully resolved. Here, auxin concentrations were measured throughout the plant prior to and during the process of tuber formation. Auxin levels increase dramatically in the stolon prior to tuberization and remain relatively high during subsequent tuber growth, suggesting a promoting role for auxin in tuber formation. Furthermore, in vitro tuberization experiments showed higher levels of tuber formation from axillary buds of explants where the auxin source (stolon tip) had been removed. This phenotype could be rescued by application of auxin on the ablated stolon tips. In addition, a synthetic strigolactone analogue applied on the basal part of the stolon resulted in fewer tubers. The experiments indicate that a system for the production and directional transport of auxin exists in stolons and acts synergistically with strigolactones to control the outgrowth of the axillary stolon buds, similar to the control of above-ground shoot branching.

Indole-3-acetic acid biosensor based on G-rich DNA labeled AuNPs as chemiluminescence probe coupling the DNA signal amplification.

A highly sensitive chemiluminescence (CL) method for detection of phytohormone indole-3-acetic acid (IAA) was developed by using G-rich DNA labeled gold nanoparticles (AuNPs) as CL probe coupling the DNA signal amplification technology. The IAA antibody was immobilized on carboxyl terminated magnetic beads (MBs). In the presence of IAA, antibody labeled AuNPs were captured by antibody functionalized MBs. The DNA on AuNPs is released by a ligand exchange process induced by the addition of DTT. The released DNA is then acted as the linker and hybridized with the capture DNA on MBs and probe DNA on AuNPs CL probe. The CL signal is obtained via the instantaneous derivatization reaction between a specific CL reagent, 3,4,5-trimethoxyl-phenylglyoxal (TMPG), and the G-rich DNA on AuNPs CL probe. IAA can be detected in the concentration range from 0.02 ng/mL to 30 ng/mL, and the limit of detection is 0.01 ng/mL.

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.

Simultaneous determination of different endogenetic plant growth regulators in common green seaweeds using dispersive liquid-liquid microextraction method.

A simple and rapid HPLC-based method was developed for simultaneous determination of major classes of plant growth regulators (PGRs) in Monostroma and different species of Ulva. The plant growth regulators determined included gibberellic acid (GA(3)), indole-3-acetic acid (IAA), abscisic acid (ABA), indole-3-butyric acid (IBA), salicylic acid and kinetin riboside (KR) and their respective elution time was 2.75, 3.3, 3.91, 4.95, 5.39 and 6.59 min. The parameters optimized for distinct separation of PGRs were mobile phase (60:40 methanol and 0.6% acetic acid in water), column temperature (35°C) and flow rate (1ml/min). This method presented an excellent linearity (0.2-100µg/ml) with limit of detection (LOD) as 0.2µg/ml for ABA, 0.5µg/ml for KR and salicylic acid, and 1µg/ml for IAA, IBA and GA(3). The precision and accuracy of the method was evaluated after inter and intra day analysis in triplicates. The effect of plant matrix was compensated after spiking and the resultant recoveries estimated were in the range of 80-120%. Each PGR thereby detected were further characterized by ESI-MS analysis. The method optimized in this study determined IBA along with IAA for the first time in the seaweed species investigated except Ulva linza where the former was not detected. In all the species studied, ABA level was detected to be the highest while kinetin riboside was the lowest. In comparison to earlier methods of PGR analysis, sample preparation and analysis time were substantially reduced while allowing determination of more classes of PGRs simultaneously.

A disposable electrochemical sensor for the determination of indole-3-acetic acid based on poly(safranine T)-reduced graphene oxide nanocomposite.

A disposable electrochemical sensor for the determination of indole-3-acetic acid (IAA) based on nanocomposites of reduced graphene oxide (rGO) and poly(safranine T) (PST) was reported. The sensor was prepared by coating a rGO film on a pre-anodized graphite electrode (AGE) through dipping-drying and electrodepositing a uniform PST layer on the rGO film. Scanning electron microscopic (SEM) and infrared spectroscopic (IR) characterizations indicated that PST-rGO formed a rough and crumpled composite film on AGE, which exhibited high sensitive response for the oxidation of IAA with 147-fold enhancement of the current signal compared with bare AGE. The voltammetric current has a good linear relationship with IAA concentration in the range 1.0×10(-7)-7.0×10(-6)M, with a low detection limit of 5.0×10(-8)M. This sensor has been applied to the determination of IAA in the extract samples of several plant leaves and the recoveries varied in the range of 97.71-103.43%.

Development of sample preparation method for auxin analysis in plants by vacuum microwave-assisted extraction combined with molecularly imprinted clean-up procedure.

A novel sample preparation method for auxin analysis in plant samples was developed by vacuum microwave-assisted extraction (VMAE) followed by molecularly imprinted clean-up procedure. The method was based on two steps. In the first one, conventional solvent extraction was replaced by VMAE for extraction of auxins from plant tissues. This step provided efficient extraction of 3-indole acetic acid (IAA) from plant with dramatically decreased extraction time, furthermore prevented auxins from degradation by creating a reduced oxygen environment under vacuum condition. In the second step, the raw extract of VMAE was further subjected to a clean-up procedure by magnetic molecularly imprinted polymer (MIP) beads. Owing to the high molecular recognition ability of the magnetic MIP beads for IAA and 3-indole-butyric acid (IBA), the two target auxins in plants can be selectively enriched and the interfering substance can be eliminated by dealing with a magnetic separation procedure. Both the VMAE and the molecularly imprinted clean-up conditions were investigated. The proposed sample preparation method was coupled with high-performance liquid chromatogram and fluorescence detection for determination of IAA and IBA in peas and rice. The detection limits obtained for IAA and IBA were 0.47 and 1.6 ng/mL and the relative standard deviation were 2.3% and 2.1%, respectively. The IAA contents in pea seeds, pea embryo, pea roots and rice seeds were determined. The recoveries were ranged from 70.0% to 85.6%. The proposed method was also applied to investigate the developmental profiles of IAA concentration in pea seeds and rice seeds during seed germination.

A high-throughput method for the quantitative analysis of auxins.

Auxin measurements in plants are critical to understanding both auxin signaling and metabolic homeostasis. The most abundant natural auxin is indole-3-acetic acid (IAA). This protocol is for the precise, high-throughput determination of free IAA in plant tissue by isotope dilution analysis using gas chromatography-mass spectrometry (GC-MS). The steps described are as follows: harvesting of plant material; amino and polymethylmethacrylate solid-phase purification followed by derivatization with diazomethane (either manual or robotic); GC-MS analysis; and data analysis. [¹³C6]IAA is the standard used. The amount of tissue required is relatively small (25 mg of fresh weight) and one can process more than 500 samples per week using an automated system. To extract eight samples, this procedure takes ∼3 h, whether performed manually or robotically. For processing more than eight samples, robotic extraction becomes substantially more time efficient, saving at least 0.5 h per additional batch of eight samples.

Heterologous expression of Arabidopsis H+-pyrophosphatase enhances salt tolerance in transgenic creeping bentgrass (Agrostis stolonifera L.).

The Arabidopsis vacuolar H(+)-pyrophosphatase (AVP1), when over-expressed in transgenic (TG) plants, regulates root and shoot development via facilitation of auxin flux, and enhances plant resistance to salt and drought stresses. Here, we report that TG perennial creeping bentgrass plants over-expressing AVP1 exhibited improved resistance to salinity than wild-type (WT) controls. Compared to WT plants, TGs grew well in the presence of 100 mm NaCl, and exhibited higher tolerance and faster recovery from damages from exposure to 200 and 300 mm NaCl. The improved performance of the TG plants was associated with higher relative water content (RWC), higher Na(+) uptake and lower solute leakage in leaf tissues, and with higher concentrations of Na(+), K(+), Cl(-) and total phosphorus in root tissues. Under salt stress, proline content was increased in both WT and TG plants, but more significantly in TGs. Moreover, TG plants exhibited greater biomass production than WT controls under both normal and elevated salinity conditions. When subjected to salt stress, fresh (FW) and dry weights (DW) of both leaves and roots decreased more significantly in WT than in TG plants. Our results demonstrated the great potential of genetic manipulation of vacuolar H(+)-pyrophosphatase expression in TG perennial species for improvement of plant abiotic stress resistance.

A high-throughput method for the quantitative analysis of indole-3-acetic acid and other auxins from plant tissue.

To investigate novel pathways involved in auxin biosynthesis, transport, metabolism, and response, we have developed a high-throughput screen for indole-3-acetic acid (IAA) levels. Historically, the quantitative analysis of IAA has been a cumbersome and time-consuming process that does not lend itself to the screening of large numbers of samples. The method described here can be performed with or without an automated liquid handler and involves purification solely by solid-phase extraction in a 96-well format, allowing the analysis of up to 96 samples per day. In preparation for quantitative analysis by selected ion monitoring-gas chromatography-mass spectrometry, the carboxylic acid moiety of IAA is derivatized by methylation. The derivatization of the IAA described here was also done in a 96-well format in which up to 96 samples can be methylated at once, minimizing the handling of the toxic reagent, diazomethane. To this end, we have designed a custom diazomethane generator that can safely withstand high flow and accommodate larger volumes. The method for IAA analysis is robust and accurate over a range of plant tissue weights and can be used to screen for and quantify other indolic auxins and compounds including indole-3-butyric acid, 4-chloro-indole-3-acetic acid, and indole-3-propionic acid.